CN107266523A - Protein purification method and fused protein and its production method containing peptide tag - Google Patents

Protein purification method and fused protein and its production method containing peptide tag Download PDF

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CN107266523A
CN107266523A CN201710199777.9A CN201710199777A CN107266523A CN 107266523 A CN107266523 A CN 107266523A CN 201710199777 A CN201710199777 A CN 201710199777A CN 107266523 A CN107266523 A CN 107266523A
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髙桥亮
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Sysmex Corp
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    • C07—ORGANIC CHEMISTRY
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    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
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    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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    • C12Y106/00—Oxidoreductases acting on NADH or NADPH (1.6)
    • C12Y106/05—Oxidoreductases acting on NADH or NADPH (1.6) with a quinone or similar compound as acceptor (1.6.5)
    • C12Y106/05002—NAD(P)H dehydrogenase (quinone) (1.6.5.2)
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    • C12Y113/00—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13)
    • C12Y113/12—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13) with incorporation of one atom of oxygen (internal monooxygenases or internal mixed function oxidases)(1.13.12)
    • C12Y113/12007—Photinus-luciferin 4-monooxygenase (ATP-hydrolysing) (1.13.12.7), i.e. firefly-luciferase
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    • C07—ORGANIC CHEMISTRY
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    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
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    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)

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Abstract

The present invention relates to protein purification method and fused protein and its production method containing peptide tag, the process and separation that are more particularly to include the sample of the fused protein of amino acid sequence of the modulation comprising the amino acid sequence containing the peptide tag containing acidic amino acid residues more than 12 residues and target protein are together contained in the protein purification method of the process of the fused protein in scrambled proteins matter and the said sample in said sample with above-mentioned fused protein.

Description

Protein purification method and fused protein and its production method containing peptide tag
【Technical field】
The present invention relates to protein purification method and fused protein and its production method containing peptide tag.
【Background technology】
By the progress of molecular biology, the weight for encoding target protein is imported to the host of Escherichia coli, yeast, cell etc. Group gene, has developed the system of great expression.In such a system, from the sample that host modulates, due to purpose The scrambled proteins matter of protein together endogenous protein containing Hosts etc., have be further purified target protein must Will.As the method for purifying target protein, for example, in Stubenrauch K, et al, Purification of a viral coat protein by an engineered polyionic sequence.J.Chromatogr.B.Biomed.Sci.Appl：737:77-84, in 2000, is disclosed to polyoma VP1 albumen 8, glutamic acid and cysteine are imported, VP1 method of protein is purified.
【The content of the invention】
【The invention technical task to be solved】
The method for purifying recombinant proteins that the present invention can easily obtain high-purity to provide is used as problem.
【Solve the technical scheme of problem】
The present invention is protein purification method, and it includes modulation and contains amino acid sequence and target protein containing peptide tag The process of the sample of the fused protein of the amino acid sequence of matter and separation together contain mixing in the sample with fused protein The process of fused protein in protein and sample, peptide tag is containing acidic amino acid residues more than 12 residues.
In addition, the present invention be the amino acid sequence containing the peptide tag containing acidic amino acid residues more than 12 residues and The fused protein of the amino acid sequence of target protein.
【Invention effect】
The high recombinant protein of purification degrees can be obtained by easy method.
【Brief description of the drawings】
【Fig. 1】It is the figure of the principle for the purification process for schematically showing present embodiment.(a) display separation circuit, (b) and (c) display target protein obtains process.
【Fig. 2】It is shown in SDS (lauryl sodium sulfate)-polyacrylamide gel electrophoresis (SDS- in embodiment 1 PAGE the figure of result).
【Fig. 3】The figure of the result for the SDS-PAGE being shown in embodiment 2.
【Fig. 4】The figure of the result for the SDS-PAGE being shown in embodiment 3.
【Fig. 5】The figure of the result for the SDS-PAGE being shown in embodiment 4.
【Fig. 6】The figure of the result for the SDS-PAGE being shown in embodiment 5.
【Fig. 7】The figure of the result for the SDS-PAGE being shown in embodiment 6.
【Fig. 8】The figure of the result for the SDS-PAGE being shown in embodiment 7.
【Fig. 9】The figure of the result for the SDS-PAGE being shown in embodiment 8.
【Figure 10】The figure of the result for the SDS-PAGE being shown in embodiment 9.
【Figure 11】The figure of the result for the SDS-PAGE being shown in embodiment 10.
【Figure 12】The figure of the result for the SDS-PAGE being shown in embodiment 11.
【Figure 13】The figure of the result for the SDS-PAGE being shown in embodiment 12.
【Figure 14】The figure of the result for the SDS-PAGE being shown in embodiment 13.
【Figure 15】The figure of the result for the SDS-PAGE being shown in comparative example 1.
【Figure 16】The figure of the result for the SDS-PAGE being shown in embodiment 14.
【Figure 17】The figure of the result for the SDS-PAGE being shown in embodiment 15.
【Figure 18】The figure of the result for the SDS-PAGE being shown in embodiment 16.
【Figure 19】The figure of the result for the SDS-PAGE being shown in embodiment 17.
【Figure 20】The figure of the result for the SDS-PAGE being shown in embodiment 18.
【Figure 21】The figure of the result for the SDS-PAGE being shown in embodiment 19.
【Figure 22】The figure of the result for the SDS-PAGE being shown in embodiment 20.
【Figure 23】The figure of the result for the SDS-PAGE being shown in embodiment 21.
【Figure 24】The figure of the result for the SDS-PAGE being shown in embodiment 22.
【Figure 25】The figure of the result for the SDS-PAGE being shown in embodiment 23.
【Figure 26】The figure of the result for the SDS-PAGE being shown in embodiment 24.
【Figure 27】The figure of the result for the SDS-PAGE being shown in embodiment 25.
【Figure 28】The figure of the result for the SDS-PAGE being shown in embodiment 26.
【Figure 29】The figure of the result for the SDS-PAGE being shown in embodiment 27.
【Figure 30】The figure of the result for the SDS-PAGE being shown in embodiment 28.
【Embodiment】
The purification process of present embodiment includes modulation containing amino acid sequence and the ammonia of target protein containing peptide tag The process (sample modulating process) of the fused protein of base acid sequence and the sample of scrambled proteins matter, and separation fused protein and The process (separation circuit) of scrambled proteins matter.Scrambled proteins matter refer to the fused proteins such as the endogenous protein of Hosts or Protein beyond target protein.
(sample modulating process)
Amino acid sequence of the fused protein containing peptide tag and target protein.As long as peptide tag containing 12 residues more than Acidic amino acid residue.Acidic amino acid residue number in peptide tag, in separation circuit, in order to scrambled proteins matter Separation become better, can further suppress the separation of causing to the isoelectric point and the pH of buffer solution by target protein etc. It is more than influence, preferably 18 residues, it is more than more preferably 24 residues, further preferably more than 30 residues, particularly preferred 36 It is more than individual residue.The upper limit of acidic amino acid residue number is not particularly limited, but from the shadow of the stereochemical structure to fused protein From the viewpoint of sound etc., below preferably 100 residues, below more preferably 70 residues.Contained acidic amino acid residue in peptide tag Can for asparagicacid residue or glutaminic acid residue either one, or two sides.Peptide tag, except acidic amino acid residue it Outside, neutral amino acid residue and/or alkaline amino acid residue can also be contained, but acidic amino acid residue number is to the overall ammonia of peptide tag The ratio of base acid residue number, in order to which the separation with scrambled proteins matter becomes better, preferably more than 20%, more preferably 60% with On.In addition, in the overall sequence of peptide tag, the amino acid sequence containing acidic amino acid residue can be acidic amino acid residue All continuous sequences, or one or more neutral amino acid residue and/or alkaline amino acid residue is between acid amino Between sour residue.For the isoelectric point (pI) of peptide tag, in order to the separation with scrambled proteins matter become it is better, can be further Suppress to the isoelectric point and the pH of buffer solution by target protein etc. cause separation influence, preferably less than 5, more preferably 4 with Under.Isoelectric point be the positive and negative in the aqueous solution ion concentration become it is equal when pH value, for example, can be by isoelectric point electrophoresis Deng measure.As the preferred concrete example of peptide tag, following (D of peptide tag 1~8 can be enumerated；Asparagicacid residue, E；Glutamic acid Residue).
<Peptide tag>
The EEEEEEDDDDDD of peptide tag 1 (DE12, SEQ ID NO：1)
The EEEEEEEEEDDDDDDDDD of peptide tag 2 (DE18, SEQ ID NO：2)
The EEEEEEEEEEEEDDDDDDDDDDDD of peptide tag 3 (DE24, SEQ ID NO：3)
The EEEEEEEEEEEEEEEDDDDDDDDDDDDDDD of peptide tag 4 (DE30, SEQ ID NO：4)
The EEEEEEEEEEEEEEEEEEDDDDDDDDDDDDDDDDDD of peptide tag 5 (DE36, SEQ ID NO：5)
The NVEGKTGNATDEEEEEEEEEEEEDDDDDDDDDDDDEDSGAEIQDDDEEGFDDEEEF DDDDD of peptide tag 6 DEHDDDDLENEENELEELEERVEARKK (DED, SEQ ID NO：6)
The DLSNVEGKTGNATDEEEEEEEEEEEEDDDDDDDDDDDDEDSGAE of peptide tag 7 (DES, SEQ ID NO：7)
The EEEEEEEEEEEEEEEEEEEEEEEE of peptide tag 8 (EO24, SEQ ID NO：8)
Target protein is not particularly limited, for example, enzyme can be enumerated, it is acceptor, interferons, interleukins class, antibody, glimmering The protein of photoprotein matter etc..The isoelectric point of target protein is not particularly limited.In general, the isoelectric point of protein be 6~ 7, but when using any protein as target, also by peptide tag is merged, can be reduced to isoelectric point can be with scrambled proteins The degree that matter is sufficiently separated.
In fused protein, peptide tag can also be combined with the N-terminal side of target protein or the either side of C-terminal side. , also can be to inserting other amino acid sequences between target protein although peptide tag can be abutted and combined with target protein in addition Row.If for example, inserting the cut-out position of protein decomposition enzyme identification between fused protein and the amino acid sequence of peptide tag The amino acid sequence of point, then cut off peptide tag from fused protein and obtain target protein and become easier to.Fused protein Isoelectric point be not particularly limited, but preferably less than 6, more preferably less than 5.Most protein are usually 6~7 due to isoelectric point, such as The isoelectric point of fruit fused protein is less than 6, and the separation of fused protein and scrambled proteins matter just becomes easier to, and can make fusion egg The purity of white matter is improved.
Sample containing fused protein and scrambled proteins matter can be by importing containing the base for encoding peptide tag to host cell The polynucleotides of the base sequence of sequence and encoding target protein, make fused protein in host cell expression to obtain.Melt The expression of hop protein matter can be carried out according to known genetic engineering method.Encode the polynucleotides and encoding target egg of peptide tag The polynucleotides of white matter method can be obtained as known to chemical synthesis etc., can be using it as template according to known gene magnification side Method is expanded.By the way that the polynucleotides and expression vector of amplification, by restriction enzyme ferment treatment, are made using appropriate DNA ligase It is combined, and can build the carrier of the expression restructuring containing each polynucleotides of coding peptide tag and target protein.For expression The structure of carrier, the base sequence of coding peptide tag can also be configured the 5 ' sides or 3 ' sides of the base sequence in encoding target protein Either side.Although the base sequence for the base sequence and encoding target protein for encoding peptide tag can abut configuration, also may be used The insertions such as the base sequence in cut-out site of encoding proteins matter catabolic enzyme identification are therebetween.As host, it is not particularly limited, can makes With Escherichia coli, yeast, insect cell, polypide, zooblast, plant cell etc..As expression vector, it is not particularly limited, can Plasmid vector, phage vector, viral vector etc. are enumerated, can suitably be selected according to the host used.In expression vector, also may be used The sequence of regulatory sequence containing replication orgin, promoter sequence, enhancer sequence etc., selection marker thing etc..Table is imported to host It can be carried out up to carrier according to host according to known method, for example, calcium phosphate method, electroporation, lipofection etc. can be enumerated.This Sample, can obtain the transformant that expression vector is imported to host cell.Obtained transformant is incubated under appropriate conditions, can make Fused protein is produced.
Make fused protein express after, to cell, tissue, polypide of host etc., as needed, extracted, crushed, The processing of centrifugation, solubilising, biological fluid extracting etc., is obtained as cell culture fluid, extract solution, homogenate, lysate, body fluid etc. Sample.In the sample, in addition to the fused protein of generation, the scrambled proteins of endogenous protein containing Hosts etc. Matter.In the modulation of sample, phosphate buffer, citrate buffer solution, acetate buffer solution, Tris buffer solutions, MOPS can be used to delay The buffer solution of fliud flushing, HEPES buffer solution etc..In these buffer solutions, containing used to obtain cushioning effect with weak acid or The salt of weak base, but in addition, the salt of sodium chloride, potassium chloride, calcium chloride etc. can be used.In this manual, buffer solution or examination The salinity of sample refer to using such cushioning effect as the salt of purpose beyond salt concentration.The salinity of sample, from fusion From the viewpoint of the stability of protein and the easiness of the adjustment in separation circuit etc., preferably more than 50mM below 500mM, More preferably more than 50mM below 250mM.The pH of other sample, the adjustment from the stability of fused protein and in separation circuit Easiness etc. from the viewpoint of, preferably less than more than 59, more preferably less than more than 68.
When obtaining sample using escherichia expression system, as expression vector, plasmid vector can be used.Carried as plasmid Body, specifically, can enumerate pET, pGEX, pCold, pMAL, pCAL etc..In expression vector, can also contain replication orgin, lac, The selection marker of T7, tac etc. promoter sequence, enhancer sequence, Ampicillin resistance gene, kalamycin resistance gene etc. The sequence of thing.Importing expression vector to Escherichia coli can be carried out by calcium phosphate method, electroporation, lipofection etc..Expression vector After importing, by using the medium culture of the thing containing selection marker, it may be selected to import the transformant of expression vector to Escherichia coli.Make After the transformant so obtained is bred under appropriate conditions, induced expression is carried out according to the promoter of selection, makes fusion egg White matter is produced.Make after fused protein expression, Escherichia coli can be carried out broken etc. and obtained containing fused protein and large intestine The sample of the scrambled proteins matter in bacillus source.
As expression system, using silkworm etc. polypide or insect cell when, transfer vector can be used.By transfer vector with After the contransduction of baculovirus DNA one is into insect cell, target gene can be inserted from homologous recombination to baculovirus DNA.Make For transfer vector, for example, pM01, pM02, pHS01, pHS02 etc. can be enumerated.Transfer vector preferably containing polyhedrin promoter, The promoter of p10 promoters etc..As insect cell, such as can enumerate Sf9, Sf21, High5, TN-368, Bm5.It is used as bar Shape virus, can enumerate such as nuclear polyhedrosis virus (NPV), specifically, can illustrate AcMNPV, BmNPV etc..By to insect Cell imports the baculovirus DNA of transfer vector and linearisation, occurs homologous recombination, obtains inserting the restructuring bar of target gene Shape virus.By being inoculated with viral solution containing recombinant baculovirus etc. to polypide or insect cell, recombinant baculovirus sense can be made Dye.The polypide of silkworm etc. can be any form of adult, pupa and larva.By making recombinate shape virus infection in polypide or insect Cell, raises or cultivates 1~7 day, can express fused protein.Make fused protein express after, it is thin to polypide or insect Born of the same parents carry out broken etc., can obtain the sample of the scrambled proteins matter containing fused protein and polypide or insect cell source.
(fused protein)
Fused protein in the present embodiment can be imported by sample modulating process described above, including to host cell Containing coding peptide tag amino acid sequence base sequence and encoding fusion protein matter amino acid sequence base sequence it is many The process of the nucleotides and production method of the process of expressed fusion protein matter is obtained in host cell.Fused protein contains The amino acid sequence and the amino acid sequence of target protein of the peptide tag of acidic amino acid residues more than 12 residues.As The fused protein of present embodiment, for example, can enumerate as the NAD of target protein (P) H dehydrogenases, the people of quinone 1 (NQ01, PI=8.91, SEQ ID NO：Or luciferase (pI=6.71, SEQ ID NO 9)：10) merged with above-mentioned peptide tag 1~8, its Between insertion protein decomposition enzyme HRV3C identification cut-out site following constructs 1~12.Will be as mesh in addition, can also illustrate Mark HRV3C (pI=8.46, SEQ the ID NO of protein：11) the following constructs 13 merged with above-mentioned peptide tag 7.
The DE12-HRV3Csite-NQ01 of construct 1 (SEQ ID NO：12)
The DE18-HRV3Csite-NQ01 of construct 2 (SEQ ID NO：13)
The DE24-HRV3Csite-NQ01 of construct 3 (SEQ ID NO：14)
The DE30-HRV3Csite-NQ01 of construct 4 (SEQ ID NO：15)
The DE36-HRV3Csite-NQ01 of construct 5 (SEQ ID NO：16)
The DED-HRV3Csite-NQ01 of construct 6 (SEQ ID NO：17)
The DES-HRV3Csite-NQ01 of construct 7 (SEQ ID NO：18)
The EO24-HRV3Csite-NQ01 of construct 8 (SEQ ID NO：19)
The NQ01-HRV3Csite-DED of construct 9 (SEQ ID NO：20)
The NQ01-HRV3Csite-DES of construct 10 (SEQ ID NO：21)
The DED-HRV3Csite- luciferases of construct 11 (SEQ ID NO：22)
The DES-HRV3Csite- luciferases of construct 12 (SEQ ID NO：23)
The DED-HRV3C of construct 13 (SEQ ID NO：24)
(separation circuit)
Sample for being contained in the fused protein obtained in sample modulating process and scrambled proteins matter, separates fusion protein Matter and scrambled proteins matter." separation fused protein and scrambled proteins matter " includes fused protein is extracted-purified, and collecting is arrived In the container different from scrambled proteins matter.Scrambled proteins matter can be substantially completely separated from, also can be by the one of scrambled proteins matter It is partially separated.By separate scrambled proteins matter part or all, can improve the purity of fused protein.
As separation means, it is not particularly limited, known separation means can be used, but in order to obtain and scrambled proteins matter Good separation, is preferably separated by ion exchange resin, more preferably by Anion exchange resin separation.It is used as anion exchange tree The ion-exchange group of fat, is not particularly limited, and quaternary ammonium (QA), quaternary ammonium ethyl (QAE), diethylamino ethyl (DEAE) can be used Deng, can specifically enumerate Hitrap HP Q (GE Healthcare companies), TOYOPEARL GigaCap Q-650M, TOYOPEARL Q-600C AR、TOYOPEARL QAE-550、TOYOPEARL DEAE-650M、TOYOPEARL SuperQ- 650M (above, Tosoh companies), Q101, DE101 (above, Mitsubishi Chemical Ind) etc.., can as the carrier of ion-exchange group Use cellulose, glucan, agarose, hydrophilic vinyl polymer etc..Make what is obtained in sample modulating process to contain fusion Protein and the sample of scrambled proteins matter make fused protein and anion exchange tree by such anion exchange resin Fat is combined.Thereafter, for example, raising the salinity of dissolution fluid, and after making scrambled proteins matter dissolution, by using high salt concentration Fused protein from ion exchange resin dissolution, can be obtained the fused protein of purifying by dissolution fluid.As making the salt of dissolution fluid The elevated method of concentration, can enumerate and periodically change salinity and make the stepwise process of target protein dissolution, continuously change Salinity and make gradient method of target protein dissolution etc..As the buffer solution used in dissolution, usable phosphate buffer, The buffer solution of citrate buffer solution, acetate buffer solution, Tris buffer solutions, MOPS buffer solutions, HEPES buffer solution etc..Buffer solution Salinity, from separated with scrambled proteins matter and the stability etc. of fused protein from the viewpoint of, be preferably set to more than 50mM, Below 2000mM scope, more preferably more than 50mM, below 1000mM scope.Particularly, if by the salinity of buffer solution More than 600mM, more preferably more than 750mM are set to, then becomes better with the separation of scrambled proteins matter.In addition, buffer solution Though pH scope is not particularly limited, from separated with scrambled proteins matter and the stability etc. of fused protein from the viewpoint of, it is excellent Select less than more than 59 scope, more preferably more than 6, less than 8 scope.
Fig. 1 is the ideograph for showing one embodiment of the present invention, and (a) is the figure for showing separation circuit.Based on this figure, say The principle of the bright separation circuit in the purification process of present embodiment.Make fused protein in host cell expression, thin Born of the same parents are crushed in the sample of liquid etc., in addition to fused protein 20, and also endogenous protein containing Hosts etc. mixes egg White matter 21A~21F.The isoelectric point of scrambled proteins matter 21 is from low to high according to A to F order.In the present embodiment, make to contain 12 The peptide tag 20b of acidic amino acid residue more than residue be incorporated into target protein 20a and make fused protein 20 etc. Electricity point reduction.The fused protein 20 that making isoelectric point reduces can be kept to high salt concentration on anion exchange resin 10.One side Face, the scrambled proteins matter of most of Hosts can not be kept in anion exchange resin 10 to the salinity of same degree.It is based on The differential separation fused protein and scrambled proteins matter of such isoelectric point.Specifically, if handing over sample contacts anion Resin 10 is changed, then the high scrambled proteins matter 21A of isoelectric point is not combined and passed through with anion exchange resin.In addition scrambled proteins Matter and fused protein 20 are combined with resin anion (R.A.) 20.If raising the salinity of dissolution fluid, mixed from isoelectric point is high Heteroproteins order dissolution (21B~D).Fused protein 20 is again by the dissolution fluid dissolution of high salt concentration.In this dissolution fraction Containing scrambled proteins the matter 21E and 21F close with the isoelectric point of fused protein 20.By so adjusting the salinity of dissolution fluid, because To separate a variety of scrambled proteins matter, the fused protein of high-purity can obtain.Further, since fused protein and scrambled proteins The difference of isoelectric point between matter is relatively large, the stepwise process of protein dissolution is also obtained good by periodically changing salinity Good separation.
(target protein acquirement process)
As described above, fused protein contained in separable sample and scrambled proteins matter, but if fused protein is also The cut-out site recognized containing protein decomposition enzyme, then, can be from by making protein decomposition enzyme act on the fused protein of separation Fused protein cuts off peptide tag, only obtains target protein.The purification process of present embodiment also may be used after separation circuit Including so using protein decomposition enzyme, peptide tag is cut off from fused protein in the solution, the process for obtaining target protein.
As the species of protein decomposition enzyme, it is not particularly limited, but for example, HRV3C, PreScission albumen can be enumerated Enzyme, factor Xa, fibrin ferment, TEV protease etc..Obtained by the way that such protein decomposition enzyme is added to for example in separation circuit To the dissolution fluid containing target protein in, can from fused protein cut off peptide tag.Reaction temperature and reaction time can bases Species of protein decomposition enzyme etc. is suitably set.Contain target protein, peptide tag and breaks down proteins in solution after the reaction Enzyme.From this solution, known separation means separation target protein can be used.As separation means, it is not particularly limited, can be used Separation means known to various chromatographies etc..It is exactly wherein, to cut off peptide tag from fused protein, then due in target protein With on peptide tag occur isoelectric point difference, separated preferably by the difference of this isoelectric point by ion exchange resin, further preferably by Anion exchange resin separation.If making to contain by the reacted target protein of protein decomposition enzyme, peptide tag and protein point The solution of solution enzyme is contacted with anion exchange resin, then cut-off peptide tag is combined with ion exchange resin, but target protein Matter is not combined and passed through with ion exchange resin.So as to which target protein can be obtained by fraction by gathering this.Containing target The salinity of the solution of protein, peptide tag and protein decomposition enzyme becomes better for the separation with peptide tag, preferably Below 500mM, more preferably below 300mM.The pH preferably more than 5, less than 9, more preferably more than 6, less than 8 of other solution.As The buffer solution used in the salinity of solution and pH adjustment, can be used phosphate buffer, citrate buffer solution, acetate buffer Liquid, Tris buffer solutions, MOPS buffer solutions, HEPES buffer solution etc..In addition, what is obtained in separation circuit contains fusion protein Scrambled proteins matter is remained in the solution fraction of matter, their isoelectric point and fused protein are relatively.Therefore, by from melting Hop protein matter cuts off peptide tag, also becomes big with the difference of the isoelectric point of the scrambled proteins matter of residual.So as to if using ion exchange Resin, then also become good with the separation of scrambled proteins matter, can obtain the target protein of higher purity.Other breaks down proteins Enzyme can be separated by known separation means, but as long as the isoelectric point of target protein and protein decomposition enzyme is variant, can by from Sub-exchange resin is easily separated both.
The isoelectric point of protein decomposition enzyme is different preferably from the isoelectric point of target protein.Thus, with breaks down proteins Digestion is broken after peptide tag, can be easily separated target protein and protein decomposition enzyme.Protein decomposition enzyme also can fusogenic peptide Label.Thus, the isoelectric point of protein decomposition enzyme can be adjusted to desired value.It is used as the peptide mark merged with protein decomposition enzyme Label, the peptide tag identical peptide tag that can be used and combined with above-mentioned fused protein.It is incorporated into the peptide of protein decomposition enzyme The amino acid sequence of the amino acid sequence of label and the peptide tag for being incorporated into fused protein can be identical, also can be different.Suitably It is and peptide tag identical amino acid sequence contained in fused protein.Thus, protein decomposition enzyme reaches with fused protein To the isoelectric point of same degree, and reach the isoelectric points different from target protein.The protein decomposition enzyme of peptide tag is merged, is Avoid and being decomposed by itself, the cut-out site preferably recognized without this protein decomposition enzyme.Use the protein point of fusion peptide tag When solving enzyme, in solution after the reaction, containing target protein, cut-off peptide tag and the breaks down proteins for merging peptide tag Enzyme.By making this solution by anion exchange resin, the protein decomposition enzyme and cut-off peptide tag one of peptide tag are merged Combined with anion exchange resin, the process without separately protein isolate matter catabolic enzyme.
The protein decomposition enzyme of peptide tag is merged, in the expression of above-mentioned fused protein, by with protein decomposition enzyme As target protein, it can obtain merging the protein decomposition enzyme of peptide tag.Fused protein and protein decomposition enzyme can be to places Dominate the carrier of the polynucleotides of the base sequence of the amino acid sequence into insertion two sides containing coding and make expression.In addition, also may be used The carrier of the polynucleotides of encoding fusion protein matter and the polynucleotides of insertion encoding proteins matter catabolic enzyme are imported to host Carrier and express it.In addition, the carrier for the polynucleotides for inserting encoding fusion protein matter can be also imported to host, to host Import after the carrier of the polynucleotides of insertion encoding proteins matter catabolic enzyme, respective single expression, mixing.Now, host can be It is of the same race, or xenogenesis, it is preferably of the same race from the point of view of processing easiness after expression etc..Merge the protein decomposition enzyme of peptide tag Isoelectric point be not particularly limited, but in order to which the separation with target protein becomes better, preferably less than 6, more preferably less than 5.
Based on Fig. 1 (b) and (c), illustrate that the target protein in the purification process of present embodiment obtains the original of process Reason.The cut-out site that fused protein 20 is recognized between target protein 20a and peptide tag 20b containing protein decomposition enzyme 22 20c.In the protein decomposition enzyme 22 of peptide tag has been merged, peptide tag 22b is combined on protein decomposition enzyme 20a.If In the solution react fused protein 20 and protein decomposition enzyme 22, then from the cut-out peptide tag of fused protein 20 20b.Anti- Should after solution in, 20a containing target protein, cut-off peptide tag 20b, merge peptide tag protein decomposition enzyme 22 and Scrambled proteins matter 21E, 21F remained in separation circuit.Because the low peptide tag 20b of isoelectric point is cut off, target protein 20a Isoelectric point is uprised compared with fused protein 20.Other scrambled proteins matter 21E and 21F is compared due to isoelectric point with fused protein 20 It is closer to, so that target protein 20a isoelectric point is higher than these.Furthermore, target protein 20a isoelectric point is also than fusion Cut-off peptide tag 20b and peptide tag 22b protein decomposition enzyme 22 are high.Therefore, if diluting this solution and making salinity Reduction, makes by anion exchange resin 10, then peptide tag 20b, protein decomposition enzyme 22 and the scrambled proteins matter being cut off 21E, 21F are combined with anion exchange resin 10, but target protein 20a is not combined and passed through.By gathering this by fraction, The target protein 20a of high-purity can be obtained.
【Embodiment】
Hereinafter, also it is described in detail for the present invention for embodiment, but the present invention is not in any way limited to these Embodiment.
【[fused protein DE12-HRV3Csite-NQ01 (the constructs 1 of embodiment 1；PI=5.84 expression) and pure Change]】
NAD (P) H dehydrogenases as target protein, the people of quinone 1 (NQ01, pI=8.91) are blended in peptide tag 1 (DE12) N-terminal side, between NQ01 and DE12 HRV3C containing protein decomposition enzyme recognize cut-out site HRV3Csite fusion egg White matter is expressed and purified.
(1) structure of pM01_DE12 carriers
Build the carrier pM01_DE12 that peptide tag DE12 is inserted to pM01 carriers.
Make encoding D E12 polynucleotides (SEQ ID NO：25 and 26) it is annealed into pair, modulation insertion subdivision.By pM01 The NheI sites of carrier (Sysmex companies) are handled with NheI (precious biotech firm), use In-Fusion HD Cloning Kits (Clone tech companies) insertion insertion subdivision.After the carrier conversion Stellar competent cells of structure, it is seeded into On LBA plates, in cultivating 16hr at 37 DEG C.From this transformant selected for ampicillin resistant transformant according to conventional methods, matter is carried out The purifying of grain.In order to confirm the base sequence of the clone selected, sequencing confirmation (the SEQ ID NO of primer 1 are used：And primer 27) 2(SEQ ID NO：28), enter performing PCR with Big Dye Terminator v3.1 (Thermo Scientific companies) and react it Afterwards, parsed with DNA sequencer (Thermo Scientific companies).Insertion subdivision, part in addition will be imported with without variation Carrier be used as pM01_DE12.
(2) pM01_DE12_HRV3Csite_NQ01 structure
Build to pM01_DE12 insertion coding NQ01 (SEQ ID NO：9) polynucleotides (SEQ ID NO：29) table Up to carrier pM01_DE12_HRV3Csite_NQ01.
Insert polynucleotides and primer 3F (the SEQ ID NO corresponding to insertion subdivision that subdivision prepares coding NQ01： And 3R (SEQ ID NO 30)：31), as PCR enzymes, using KOD-Plus- (company is spun by Japan), expanded with following PCR conditions. The PCR primer of amplification is subjected to electrophoresis with 1.0% (w/v) agarose electrophoresis, by the part consistent with the length for inserting subdivision Cut out from gel and purify and modulate insertion subdivision.By the SmaI sites of pM01_DE12 carriers with SmaI (precious biotech firm) Processing, uses In-Fusion HD Cloning Kits (Clone tech companies) insertion insertion subdivision.Turned with the carrier of structure Change after Stellar competent cells (Clone tech companies), be seeded on LBA plates.In cultivating 16hr at 37 DEG C, from this turn Change body selected for ampicillin resistant transformant according to conventional methods, carry out the purifying of plasmid.In order to confirm the alkali of the clone selected Basic sequence, using the primer 1 and 2 of sequencing confirmation, with Big Dye Terminator v3.1, (Thermo Scientific are public Department) enter after performing PCR reaction, parsed with DNA sequencer (Thermo Scientific companies).To be imported with insertion subdivision, In addition carrier of the part without variation is used as pM01_DE12_HRV3Csite_NQ01.
<PCR conditions>
Reaction 1：94.0 DEG C, 2 minutes；Reaction 2：94.0 DEG C, 15 seconds；Reaction 3：52.0 DEG C, 30 seconds；Reaction 4：68.0 DEG C, per 1kbp1 minutes；Reaction 5：Reaction 2~reaction 4 is repeated 30 times
(3) making of recombinant baculovirus and fused protein DE12-HRV3Csite-NQ01 expression
PM01_DE12_HRV3Csite_NQ01 and baculovirus DNA are imported to silkworm culture cell (BmN cells), disease is carried out The homologous recombination of poison.This culture cell culture 6 days is reclaimed after recombinant virus, to the pupa inoculation MilliQ water dilution 50 of silkworm Viral solution again, is incubated 6 days.
(4) DE12-HRV3Csite-NQ01 purifying
After incubation, the pupa of silkworm, buffer solution (the 20mM Tris-HCl (pH 8.0), 150mM of every 1 plus 50mL are reclaimed NaCl, 1 entirely without EDTA (1tablet Complete EDTA free) (Roche), phenyltiourea) homogenate, to molten Liquid is centrifuged after (8,000g, 10 minute), and supernatant fraction is filtered with 0.8 μM of filtering agent, filtrate is reclaimed.By the filtrate of recovery Be loaded into ion exchange resin (Hitrap Q HP (1mL)), using mixing 20mM Tris-HCl (pH 8.0,150mM NaCl, Buffer A) and 20mM Tris-HCl (pH 8.0,1000mM NaCl, buffer B) solution purification.Each reclaim only makes first Buffer A (NaCl concentration 150mM) flows through ion exchange resin (EL0), uses buffer A：The 3 of buffer B:1 mixed solution (NaCl concentration 363mM) dissolution (EL1), use buffer A：The 1 of buffer B:1 mixed solution (NaCl concentration 575mM) dissolution (EL2) buffer A, is used：The 1 of buffer B:3 mixed solution (NaCl concentration 788mM) dissolutions (EL3), finally only use buffer B The fraction of (NaCl concentration 1000mM) dissolution (EL4).
(5) DE12-HRV3Csite-NQ01 detection
SDS (lauryl sodium sulfate)-polyacrylamide gel electrophoresis (SDS-PAGE) using 5-20% gradient gels and Electrophoretic apparatus (APRO Science companies) is implemented.Sample and TrisSDS β-ME sample treatment liquids (COSMO-BIO companies) are with 1: 1 Capacity Ratio is mixed, and at 100 DEG C, after processing 3 minutes, 5 μ L are loaded into gel.As molecular weight marker, by BlueStar (Japanese Genetics companies) is loaded into 2.5 μ L gels.By the gel of load sample with voltage 400V electrophoresis 14 minutes after, make It is transferred to transfer Turbo blotting systems (BIO-RAD companies) on film.The film of transfer is arranged at i-Bind systems (Thermo Fisher Scientific companies), implement antigen-antibody reaction.In antigen-antibody reaction, anti-FLAG (registration mark) M2- Peroxidase (HRP) antibody (Merck companies) or anti-NQ01 antibody (Cell signaling companies) and anti-IgG (H+L Chain) (Mouse) pAb-HRP (Beckman companies) dilutes 2000 times and used, and the HRP reagents of detection use Luminata Forte (Merck companies), detection machine use Gel Doc XR+ systems (BIO-RAD companies).
After implementation western blot, by the film used GelCode Blue Stain Reagent (Thermo Fisher Scientific companies) dyeing, after making extra dyeing-decolorzing, using Gel Doc XR+ systems (BIO-RAD companies) to figure As being photographed.As a result it is shown in Fig. 2.In fig. 2, M is that molecular weight marker, swimming lane 1 are that homogenate solution, swimming lane 2 are centrifugations Supernatant fraction afterwards, swimming lane 3 be centrifugation after pellet fraction, 4~swimming lane of swimming lane 7 be ion exchange resin by fraction 1~ 4th, it by the dissolution fraction (EL0) of buffer A (NaCl concentration 150mM), swimming lane 9 is by buffer A that swimming lane 8, which is,：The 3 of buffer B: The dissolution fraction (EL1) of 1 mixed solution (NaCl concentration 363mM), swimming lane 10 are by buffer A：The 1 of buffer B:1 mixing is molten The dissolution fraction (EL2) of liquid (NaCl concentration 575mM), swimming lane 11 are, by buffer A：The 1 of buffer B:3 mixed solution (NaCl Concentration 788mM) solution fraction (EL3), swimming lane 12 be dissolution fraction (EL4) by buffer B (NaCl concentration 1000mM) Electrophoresis result.The band containing target protein is represented with the part of dotted line.The explanation of each swimming lane is led in Fig. 3~15 With.
【[fused protein DE18-HRV3Csite-NQ01 (the constructs 2 of embodiment 2；PI=5.03 expression) and pure Change]】
The N-terminal side of the NQ01 as target protein is blended in peptide tag 2 (DE18), is contained between NQ01 and DE18 HRV3Csite fused protein is expressed and purified.
Encoding D E18 (SEQ IDNO are used except replacing coding peptide tag DE12 polynucleotides：2) polynucleotides (SEQ ID NO：32 and 33) modulation insertion subdivision outside, similarly to Example 1 build pM01_DE18 carriers.
Again similarly to Example 1, after structure pM01_DE18_HRV3Csite_NQ01, making recombinant baculovirus, Carry out fused protein DE18-HRV3Csite-NQ01 expression, purifying and detection.As a result it is shown in Fig. 3.
【[fused protein DE24-HRV3Csite-NQ01 (the constructs 3 of embodiment 3；PI=4.78 expression) and pure Change]】
The N-terminal side of the NQ01 as target protein is blended in peptide tag 3 (DE24), is contained between NQ01 and DE24 HRV3Csite fused protein is expressed and purified.
(1) structure of pM01_DE24 carriers
The carrier pM01_DE24 that coding peptide tag DE24 polynucleotides are inserted to pM01 carriers is built as the following.
Prepare coding peptide tag 6 (DED, SEQ ID NO：6) polynucleotides (SEQ ID NO：34) and corresponding to insertion Primer 4F (the SEQ ID NO of subdivision：And 4R (SEQ ID NO 35)：36), as PCR enzymes, using KOD-Plus-, (Japan is spun Company), expanded with PCR conditions same as Example 1.The PCR primer of amplification is carried out with 1.0% (w/v) agarose electrophoresis Electrophoresis, the part consistent with the length for inserting subdivision is cut out from gel, is purified and is modulated insertion subdivision.By pM01 carriers The SmaI sites of (Sysmex companies) are handled with SmaI (precious biotech firm), use In-Fusion HD Cloning Kits (Clone Tech companies) insertion insertion subdivision.With the carrier of structure conversion Stellar competent cells (Clone tech companies) it Afterwards, it is seeded on LBA plates.In cultivating 16hr at 37 DEG C, from this transformant selected for ampicillin resistant transformants according to conventional methods Body, carries out the purifying of plasmid.In order to confirm the base sequence of the clone selected, using the primer 1 and 2 of sequencing confirmation, Big is used Dye Terminator v3.1 (Thermo Scientific companies) enter after performing PCR reaction, with DNA sequencer (Thermo Scientific companies) parsing.Insertion subdivision, carrier of the part in addition without variation will be imported with and be used as pM01_DE24.
(2) similarly to Example 1, after structure pM01_DE24_HRV3Csite_NQ01, making recombinant baculovirus, Carry out fused protein DE24-HRV3Csite-NQ01 expression, purifying and detection.As a result it is shown in Fig. 4.
【[fused protein DE30-HRV3Csite-NQ01 (the constructs 4 of embodiment 4；PI=4.61 expression) and pure Change]】
The N-terminal side of the NQ01 as target protein is blended in peptide tag 4 (DE30), is contained between NQ01 and DE30 HRV3Csite fused protein is expressed and purified.
Encoding D E30 (SEQ ID NO are used except replacing coding peptide tag DE12 polynucleotides：4) polynucleotides (SEQ ID NO：37 and 38) modulation insertion subdivision outside, similarly to Example 1 build pM01_DE30 carriers.
Again similarly to Example 1, after structure pM01_DE30_HRV3Csite_NQ01, making recombinant baculovirus, Carry out fused protein DE30-HRV3Csite-NQ01 expression, purifying and detection.As a result it is shown in Fig. 5.
【[fused protein DE36-HRV3Csite-NQ01 (the constructs 5 of embodiment 5；PI=4.49 expression) and pure Change]】
The N-terminal side of the NQ01 as target protein is blended in peptide tag 5 (DE36), is contained between NQ01 and DE36 HRV3Csite fused protein is expressed and purified.
Encoding D E36 (SEQ ID NO are used except replacing coding peptide tag DE12 polynucleotides：5) polynucleotides (SEQ ID NO：39 and 40) modulation insertion subdivision outside, similarly to Example 1 build pM01_DE36 carriers.
Again similarly to Example 1, after structure pM01_DE36_HRV3Csite_NQ01, making recombinant baculovirus, Carry out fused protein DE36-HRV3Csite-NQ01 expression, purifying and detection.As a result it is shown in Fig. 6.
【[fused protein DED-HRV3Csite-NQ01 (the constructs 6 of embodiment 6；PI=4.26 expression and purifying)]】
The N-terminal side of the NQ01 as target protein is blended in peptide tag 6 (DED), is contained between NQ01 and DED HRV3Csite fused protein is expressed and purified.
(1) structure of pHS01_DED carriers
To pHS01 carriers insertion peptide tag DED (SEQ ID NO：6) carrier pHS01_DED is built as the following.
Prepare coding peptide tag DED polynucleotides (SEQ ID NO：34) and corresponding to the primer 5F of insertion subdivision (SEQ ID NO：And 5R (SEQ ID NO 41)：42), as PCR enzymes, using KOD-Plus- (Japan spin company), with implementation The identical PCR conditions of example 1 are expanded.The PCR primer of amplification is subjected to electrophoresis with 1.0% (w/v) agarose electrophoresis, will be with intron The consistent part of partial length is cut out from gel, is purified and is modulated insertion subdivision.By pHS01 carriers (Sysmex companies) NheI sites are handled with NheI (precious biotech firm), are inserted using In-Fusion HD Cloning Kits (Clone tech companies) Insert subdivision.After the carrier conversion Stellar competent cells of structure, it is seeded on LBA plates.In culture at 37 DEG C 16hr, from this transformant selected for ampicillin resistant transformant according to conventional methods, carries out the purifying of plasmid.In order to confirm to select Clone base sequence, using sequencing confirmation primer 1 and 2, with Big Dye Terminator v3.1 (Thermo Scientific companies) enter after performing PCR reaction, parsed with DNA sequencer (Thermo Scientific companies).It will import There is the insertion carrier of subdivision, part in addition without variation as pHS01_DED.
(2) HS01_DED_HRV3Csite_NQ01 is built similarly to Example 1, is made after recombinant baculovirus, is entered Row fused protein DED-HRV3Csite-NQ01 expression, purifying and detection.As a result it is shown in Fig. 7.
【[fused protein DES-HRV3Csite-NQ01 (the constructs 7 of embodiment 7；PI=4.55 expression and purifying)]】
The N-terminal side of the NQ01 as target protein is blended in peptide tag 7 (DES), is contained between NQ01 and DES HRV3Csite fused protein is expressed and purified.
Except as the primer corresponding to insertion subdivision, using primer 6F (SEQ ID NO：And 6R (SEQ ID 43) NO：44) outside, similarly to Example 6, pHS01_DES is built.
PHS01_DES_HRV3Csite_NQ01 is built similarly to Example 1, is made after recombinant baculovirus, is carried out Fused protein DES-HRV3Csite-NQ01 expression, purifying and detection.As a result it is shown in Fig. 8.
【[fused protein EO24-HRV3Csite-NQ01 (the constructs 8 of embodiment 8；PI=4.73 expression) and pure Change]】
The N-terminal side of the NQ01 as target protein is blended in peptide tag 8 (EO24), is contained between NQ01 and EO24 HRV3Csite fused protein is expressed and purified.
(1) pHS01_EO24 structure
Make encoded peptide label E O24 polynucleotides (SEQ ID NO：45 and 46) it is annealed into pair, modulation insertion subdivision. The NheI sites of pHS01 carriers (Sysmex companies) are handled with NheI (precious biotech firm), cloned using In-Fusion HD Kit (Clone tech companies) is inserted into insertion subdivision.Stellar competent cells are converted with the carrier of structure After (Clone tech companies), it is seeded on LBA plates.In cultivating 16hr at 37 DEG C, chosen according to conventional methods from this transformant Ampicillin resistant transformant is selected, the purifying of plasmid is carried out.In order to confirm the base sequence of the clone selected, confirmed using sequencing Primer 1 and 2, is entered after performing PCR reaction with Big Dye Terminator v3.1 (Thermo Scientific companies), Parsed with DNA sequencer (Thermo Scientific companies).Insertion subdivision, part in addition will be imported with without variation Carrier is used as pHS01_EO24.
(2) pHS01_EO24_HRV3Csite_NQ01 is built similarly to Example 1, after making recombinant baculovirus, Carry out fused protein EO24-HRV3Csite-NQ01 expression, purifying and detection.As a result it is shown in Fig. 9.
【[fused protein NQ01-HRV3Csite-DED (the constructs 9 of embodiment 9；PI=4.26 expression and purifying)]】
The C-terminal side as the NQ01 of target protein is blended in peptide tag DED, HRV3Csite is contained between NQ01 and DED Fused protein expressed and purified.
(1) structure of pHS02_DED carriers
The carrier pHS02_DE12 that coding peptide tag DED polypeptide is inserted to pHS02 carriers is built as the following.
Prepare coding peptide tag DED polynucleotides (SEQ ID NO：34) and corresponding to the primer 7F of insertion subdivision (SEQ ID NO：And 7R (SEQ ID NO 47)：48), as PCR enzymes, using KOD-Plus- (Japan spin company), with implementation The identical PCR conditions of example 1 are expanded.The PCR primer of amplification is subjected to electrophoresis with 1.0% (w/v) agarose electrophoresis, will be with intron The consistent part of partial length is cut out from gel, is purified and is modulated insertion subdivision.By pHS02 carriers (Sysmex companies) NheI sites are handled with NheI (precious biotech firm), are inserted using In-Fusion HD Cloning Kits (Clone tech companies) Insert subdivision.After the carrier conversion Stellar competent cells (Clone tech companies) of structure, LBA plates are seeded into On.In cultivating 16hr at 37 DEG C, from this transformant selected for ampicillin resistant transformant according to conventional methods, the pure of plasmid is carried out Change.In order to confirm the base sequence of the clone selected, using the primer 1 and 2 of sequencing confirmation, Big DyeTerminator are used V3.1 (Thermo Scientific companies) enters after performing PCR reaction, with DNA sequencer (Thermo Scientific companies) Parsing.Insertion subdivision, carrier of the part in addition without variation will be imported with and be used as pHS02_DED.
(2) except as the primer corresponding to insertion subdivision, using primer 8F (SEQ ID NO：And 8R (SEQ ID 49) NO：50) outside, pHS02_NQ01_HRV3Csite_DED is built similarly to Example 1.
(3) recombinant baculovirus is also made similarly to Example 1, carries out fused protein NQ01-HRV3Csite-DED Expression, purifying and detect.As a result it is shown in Figure 10.
【[fused protein NQ01-HRV3Csite-DES (the constructs 10 of embodiment 10；PI=4.55 expression) and pure Change]】
The C-terminal side as the NQ01 of target protein is blended in peptide tag DES, HRV3Csite is contained between NQ01 and DES Fused protein expressed and purified.
(1) except as primer, using 9F (SEQ ID NO：And 9R (SEQ ID NO 51)：52) outside, with embodiment 9 Similarly, pHS02_DES is built.
(2) pHS01_NQ01_HRV3Csite_DES is built similarly to Example 9.
(3) recombinant baculovirus is made similarly to Example 1, carries out fused protein NQ01-HRV3Csite-DES's Expression, purifying and detection.As a result it is shown in Figure 11.
【[fused protein DED-HRV3Csite-Luciferase (the constructs 11 of embodiment 11；PI=4.47 expression) And purifying]】
(1) structure of pHS01_DED carriers
PHS01_DED is built similarly to Example 6.
(2) structure of pHS01_DED_HRV3Csite_ luciferases
Prepare polynucleotides (the SEQ ID NO of Encoding Luciferase：53) and corresponding to the primer 10F of insertion subdivision (SEQ ID NO：And 10R (SEQ ID NO 54)：55), as PCR enzymes, using KOD-Plus- (Japan spin company), with reality Apply the amplification of the identical PCR conditions of example 1.The PCR primer of amplification is subjected to electrophoresis with 1.0% (w/v) agarose electrophoresis, will be with insertion The part that the length of subdivision is consistent is cut out from gel, is purified and is modulated insertion subdivision.By the SmaI positions of pHS01_DED carriers Point is handled with SmaI (precious biotech firm), and intron is inserted using In-Fusion HD Cloning Kits (Clone tech companies) Part.After the carrier conversion Stellar competent cells (Clone tech companies) of structure, it is seeded on LBA plates.In 37 16hr is cultivated at DEG C, from this transformant selected for ampicillin resistant transformant according to conventional methods, the purifying of plasmid is carried out.In order to The base sequence of clone selected is confirmed, using the primer 1 and 2 of sequencing confirmation, with Big Dye Terminator v3.1 (Thermo Scientific companies) enters after performing PCR reaction, is solved with DNA sequencer (Thermo Scientific companies) Analysis.Insertion subdivision, carrier of the part in addition without variation will be imported with and be used as pHS01_DED_HRV3Csite_ luciferases. (3) recombinant baculovirus is made similarly to Example 1, carries out the table of fused protein DED-HRV3Csite- luciferases Reach, purify and detect.As a result it is shown in Figure 12.
【[the fused protein DES-HRV3Csite- luciferase (constructs 12 of embodiment 12；PI=4.75 expression) and Purifying]】
In addition to replacing pHS01_DED carriers and using the pHS01_DES carriers built in embodiment 7, with implementation Example 11 similarly builds pHS02_DES_HRV3Csite_ luciferases.
Similarly to Example 1, recombinant baculovirus is made, fused protein DES-HRV3Csite- luciferases are carried out Expression, purifying and detect.As a result it is shown in Figure 13.
【[fused protein DED-HRV3C (the constructs 13 of embodiment 13；PI=4.75 expression and purifying)]】
(1) pET17b_DED_HRV3C structure
Prepare coding peptide tag DED polynucleotides (SEQ ID NO：34) HRV3C polynucleotides (SEQ ID, are encoded NO：56) and corresponding to primer 11F and 11R (the SEQ ID NO of insertion subdivision：57 and 58, DED) and primer 12F and 12R (SEQ ID NO：59 and 60, HRV3C), as PCR enzymes, using KOD-Plus- (Japan spin company), with same as Example 1 PCR conditions amplification.The PCR primer of amplification is subjected to electrophoresis with 1.0% (w/v) agarose electrophoresis, by with inserting subdivision The consistent part of length is cut out from gel, is purified and is modulated insertion subdivision.By the SmaI positions of pHS01 carriers (Sysmex companies) Point and NheI sites are handled with SmaI (precious biotech firm) and NheI (precious biotech firm), use In-Fusion HD cloning kits Box (Clone tech companies) inserts 2 insertion subdivisions.With the carrier conversion Stellar competent cells (Clone of structure Tech companies) after, it is seeded on LBA plates.In cultivating 16hr at 37 DEG C, ammonia benzyl west is selected according to conventional methods from this transformant Woods resistant transformant, carries out the purifying of plasmid.In order to confirm the base sequence of the clone selected, the primer of sequencing confirmation is used 1 and 2, entered with Big Dye Terminator v3.1 (Thermo Scientific companies) after performing PCR reaction, surveyed with DNA Sequence instrument (Thermo Scientific companies) is parsed.To be imported with insertion subdivision, carrier of the part in addition without variation as pHS01_DED_HRV3C。
Next, modulation pET17b intron.Using pHS01_DED_HRV3C as template, prepare primer 13F (SEQ ID NO：And 13R (SEQ ID NO 61)：62), as PCR enzymes, using KOD-Plus- (Japan spin company), with embodiment 1 Identical PCR conditions are expanded.The PCR primer of amplification is subjected to electrophoresis with 1.0% (w/v) agarose electrophoresis, will be with insertion sub-portion Point the consistent part of length cut out from gel, purifying and modulate insertion subdivision.By pET17b carriers (Novagene companies) NdeI sites with NdeI (precious biotech firm) handle, use In-Fusion HD Cloning Kits (Clone tech companies). After the carrier conversion Stellar competent cells (Clone tech companies) of structure, it is seeded on LBA plates.In at 37 DEG C 16hr is cultivated, from this transformant selected for ampicillin resistant transformant according to conventional methods, the purifying of plasmid is carried out.In order to confirm The base sequence of the clone selected, uses (the SEQ ID NO of primer 14 of sequencing confirmation：And 15 (SEQ ID NO 63)：64), Entered with Big Dye Terminator v3.1 (Thermo Scientific companies) after performing PCR reaction, use DNA sequencer (Thermo Scientific companies) parses.To be imported with insertion subdivision, carrier of the part in addition without variation as pET17b_DED_HRV3C。
(2) DED-HRV3C expression and purifying
Escherichia coli are converted using pET17b_DED_HRV3C, by the LB cultures of the 1.5L containing ampicillin based on 37 DEG C of trainings Support, be cooled near turbidity OD=0.6 after 15 DEG C, add the expression of IPTG inducible proteins.After 16 hours, with centrifugation (8, 000g 15 minutes) collection bacterium, in freezing at -80 DEG C.To Escherichia coli addition 120mL buffer solutions (the 20mM Tris-HCl of freezing (pH 8.0), 150mM NaCl, 1, entirely without EDTA (1tablet Complete EDTA free) (Roche), is used ultrasonic wave It is broken.Broken is centrifuged to solution after (15,000g × 30 minutes), and supernatant fraction is filtered with 0.8 μM of filtering agent, reclaimed Filtrate.Filtrate is loaded into ion exchange resin (Hitrap Q HP (1mL)), using mixing 20mM Tris-HCl (pH 8.0, 150mM NaCl, buffer A) and 20mM Tris-HCl (pH 8.0,1000mM NaCl, buffer B) solution purification.Each time Receipts only make buffer A (NaCl concentration 150mM) flow through ion exchange resin (EL0), use buffer A first：The 3 of buffer B:1 Mixed solution (NaCl concentration 363mM) dissolution (EL1), use buffer A：The 1 of buffer B:1 mixed solution (NaCl concentration 575mM) dissolution (EL2), use buffer A：The 1 of buffer B:3 mixed solution (NaCl concentration 788mM) dissolutions (EL3), only finally With the fraction of buffer B (NaCl concentration 1000mM) dissolution (EL4).
(3) detection fusion protein similarly to Example 1.As a result it is shown in Figure 14.In fig. 14, M is molecular weight markers Thing, swimming lane 1 be homogenate solution, swimming lane 2 be supernatant fraction after centrifugation, swimming lane 3 be pellet fraction after centrifugation, swimming lane 4~ Swimming lane 7 be ion exchange resin by fraction 1~4, swimming lane 8 is the dissolution fraction by buffer A (NaCl concentration 150mM) (EL0), swimming lane 9 is by buffer A：The 3 of buffer B:The dissolution fraction (EL1) of 1 mixed solution (NaCl concentration 363mM), swimming Road 10 is by buffer A：The 1 of buffer B:The dissolution fraction (EL2) of 1 mixed solution (NaCl concentration 575mM), swimming lane 11 be by Buffer A：The 1 of buffer B:The solution fraction (EL3) of 3 mixed solutions (NaCl concentration 788mM), swimming lane 12 are by buffer B The electrophoresis result of the dissolution fraction (EL4) of (NaCl concentration 1000mM).
【Comparative example 1 [fused protein DE6-HRV3Csite-NQ01 (pI=6.4) expression and purifying]】
The N-terminal side of the NQ01 as target protein is blended in peptide tag DE6, HRV3Csite is contained between NQ01 and DE6 Fused protein expressed and purified.
Encoding D E6 (EEEDDD, SEQ ID NO are used except replacing coding peptide tag DE12 polynucleotides：65) Polynucleotides (SEQ ID NO：66 and 67) modulation insertion subdivision outside, similarly to Example 1 build pM01_DE6 carriers.
Build pM01_DE6_HRV3Csite_NQ01 similarly to Example 1 again, make after recombinant baculovirus, enter Row fused protein DE6-HRV3Csite-NQ01 expression, purifying and detection.As a result it is shown in Figure 15.
(for embodiment 1~13 and the result of comparative example 1)
In comparative example 1 (DE6-HRV3Csite-NQ01, pI=6.4), divide in all fractions containing fused protein From insufficient.In contrast to this, good separation is shown in embodiment 1~13.Especially in conjunction with acidity more than 18 residues The fused protein of the peptide tag of amino acid residue is held in ion exchange resin to more than 500mM high salt concentration, with mixing The separation of protein becomes better (embodiment 2~13).In addition, 3 kinds of target proteins it is any in obtained good Good separation (embodiment 6~7,11~13).In addition, peptide tag, as acidic amino acid residue, no matter containing asparagicacid residue And glutaminic acid residue this two side, still only show equal good separation (embodiment 3,8) containing either one.The combination of peptide tag The either side of the position either N-terminal side of target protein or C-terminal side, equal good separation (embodiment 6~7,9~10).
【Embodiment 14 [separates target protein NQ01 and detection] from fused protein DE12-HRV3Csite-NQ01】
Protein decomposition enzyme DED-HRV3C is set to react on DE12-HRV3Csite-NQ01 and from DE12-HRV3Csite- NQ01 cuts off DE12.
The fraction reclaimed in embodiment 13 of its 1/10th capacity is mixed to the fraction EL2 reclaimed in embodiment 1 EL3, in standing at 4 DEG C.After 16 hours, 20mM Tris-HCl (pH 8.0) are added to this reaction solution and to be diluted to NaCl dense About 250mM is spent, is loaded into 20mM Tris-HCl (pH 8.0), the ion exchange resin (Hitrap of 150mM NaCl equilibratings Q HP (1mL)) on.It is each to reclaim by fraction (Fr.1-6 (NaCl concentration about 250mM)), finally only use buffer B (NaCl Concentration 1000mM) dissolution (Fr.7-10) fraction.
SDS-PAGE is implemented using 5-20% gradient gels and electrophoretic apparatus (APRO Science companies).By each of recovery Fraction is with TrisSDS β-ME sample treatment liquids (COSMO-BIO companies) with 1:1 Capacity Ratio is mixed, at 100 DEG C, processing 3 minutes it Afterwards, 5 μ L are loaded into gel.As molecular weight marker, BlueStar (Japanese Genetics companies) is loaded into 2.5 μ L and coagulated Glue.By the gel of load sample with voltage 400V electrophoresis 14 minutes after, with GelCode Blue Stain Reagent (Thermo Fisher Scientific companies) dyes, and makes after extra dyeing-decolorzing, uses Gel Doc XR+ systems (BIO-RAD companies) photographs to image.As a result it is shown in Figure 16.In figure 16, M is that molecular weight marker, swimming lane 1 are EL2 (embodiment 1,8) or EL3 (embodiment 2~7,11~12), swimming lane 2 are that the EL3 of embodiment 13, swimming lane 3 are embodiments 1~12 EL2 or EL3 and the EL3 of embodiment 13 mixed reaction solution, 4~swimming lane of swimming lane 8 be ion exchange resin by fraction 1~5, Swimming lane 9~13 is by the electrophoresis result of the dissolution fraction 1~5 of buffer B.The explanation of each swimming lane is general in Figure 17~25.
【Embodiment 15】
In addition to replacing the EL2 of embodiment 1 and using the EL2 of embodiment 2, target egg is detected similarly to Example 14 White matter NQ01.As a result it is shown in Figure 17.
【Embodiment 16】
In addition to replacing the EL2 of embodiment 1 and using the EL3 of embodiment 3, target egg is detected similarly to Example 14 White matter NQ01.As a result it is shown in Figure 18.
【Embodiment 17】
In addition to replacing the EL2 of embodiment 1 and using the EL3 of embodiment 4, target egg is detected similarly to Example 14 White matter NQ01.As a result it is shown in Figure 19.
【Embodiment 18】
In addition to replacing the EL2 of embodiment 1 and using the EL3 of embodiment 5, target egg is detected similarly to Example 14 White matter NQ01.As a result it is shown in Figure 20.
【Embodiment 19】
In addition to replacing the EL2 of embodiment 1 and using the EL3 of embodiment 6, target egg is detected similarly to Example 14 White matter NQ01.As a result it is shown in Figure 21.
【Embodiment 20】
In addition to replacing the EL2 of embodiment 1 and using the EL3 of embodiment 7, target egg is detected similarly to Example 14 White matter NQ01.As a result it is shown in Figure 22.
【Embodiment 21】
In addition to replacing the EL2 of embodiment 1 and using the EL2 of embodiment 8, target egg is detected similarly to Example 14 White matter NQ01.As a result it is shown in Figure 23.
【Embodiment 22】
In addition to replacing the EL2 of embodiment 1 and using the EL3 of embodiment 11, target is detected similarly to Example 14 Protein luciferase.As a result it is shown in Figure 24.
【Embodiment 23】
In addition to replacing the EL2 of embodiment 1 and using the EL3 of embodiment 12, target is detected similarly to Example 14 Protein luciferase.As a result it is shown in Figure 25.
(for the result of embodiment 14~23)
Shown from the result of embodiment 14~23, by making protein decomposition enzyme act on fused protein, can the moon from Passing through for subtree fat obtains target protein NQ01 or luciferase in fraction.
【Embodiment 24 [evaluation of influences of the pH of buffer solution to separation]】
Build similarly to Example 6, pHS01_DED_HRV3Csite_NQ01.
PHS01_DED_HRV3Csite_NQ01 and baculovirus DNA are imported to silkworm culture cell (BmN cells), disease is carried out The homologous recombination of poison.This culture cell culture 6 days is reclaimed after recombinant virus, to the pupa inoculation MilliQ water dilution 50 of silkworm Viral solution again, is incubated 6 days.Reclaim the pupa of silkworm, buffer solution (the 20mM PIPES (pH6.1), 150mM of every 1 plus 50mL NaCl, 1 entirely without EDTA (1tablet Complete EDTA free) (Roche), phenyltiourea) and be homogenized, its Solution is centrifuged after (8,000g, 10 minute), supernatant fraction is filtered with 0.8 μM of filtering agent, filtrate is reclaimed.By filtrate plus Ion exchange resin (Hitrap Q HP (1mL)) is downloaded to, mixing 20mM PIPES (pH6.1,150mM NaCl, buffer solution is used C) and 20mM PIPES (pH6.1,1000mM NaCl, buffer solution D) solution purification.Each reclaim only makes buffer solution C first (NaCl concentration 150mM) flows through ion exchange resin (EL0), uses buffer solution C：Buffer solution D mixing ratio 3：1 (NaCl concentration Solution dissolution (EL1) 363mM), use buffer solution C：Buffer solution D mixing ratio 1:1 (NaCl concentration 575mM) solution dissolution (EL2) buffer solution C, is used：Buffer solution D mixing ratio 1:3 (NaCl concentration 788mM) solution dissolution (EL3), finally only with buffering The fraction of liquid D (NaCl concentration 1000mM) dissolution (EL4).
DED-HRV3Csite-NQ01 is detected similarly to Example 1.As a result it is shown in Figure 26.In fig. 26, M is molecular weight Mark, swimming lane 1 are that homogenate solution, swimming lane 2 are that supernatant fraction after centrifugation, swimming lane 3 are pellet fraction, swimming lanes after centrifugation 4~swimming lane 7 be ion exchange resin by fraction 1~4, swimming lane 8 be by buffer solution C (NaCl concentration 150mM) dissolution level It is by buffer solution C to divide (EL0), swimming lane 9：The 3 of buffer solution D:The dissolution fraction (EL1) of 1 mixed solution (NaCl concentration 363mM), Swimming lane 10 is by buffer solution C：The 1 of buffer solution D:The dissolution fraction (EL2) of 1 mixed solution (NaCl concentration 575mM), swimming lane 11 are By buffer solution C：The 1 of buffer solution D:The solution fraction (EL3) of 3 mixed solutions (NaCl concentration 788mM), swimming lane 12 are by buffer solution The electrophoresis result of D (NaCl concentration 1000mM) dissolution fraction (EL4).The explanation of each swimming lane is general in Figure 27~28.
【Embodiment 25】
PHS01_DES_HRV3Csite_NQ01 is built similarly to Example 7.
Express, purify and detection DES-HRV3Csite-NQ01 similarly to Example 24 again.As a result it is shown in Figure 27.
【Embodiment 26】
PHS01_DED_HRV3Csite_ luciferases are built similarly to Example 11.
Express, purify and detection DED-HRV3Csite- luciferases similarly to Example 24 again.As a result it is shown in Figure 28.
【Embodiment 27】
PHS01_DES_HRV3Csite_NQ01 is built similarly to Example 7.
Escherichia coli are converted using pHS01_DES_HRV3Csite_NQ01, by the LB culture mediums of the 1.5L containing ampicillin In 37 DEG C of cultures, it is cooled near turbidity OD=0.6 after 15 DEG C, adds the expression of IPTG inducible proteins.After 16 hours, use (8,000g 15 minutes) collection bacterium of centrifugation, in freezing at -80 DEG C.To the Escherichia coli addition 120mL buffer solutions (20mM of freezing PIPES (pH6.1), 150mM NaCl, 1 entirely without EDTA (1tablet Complete EDTA free) (Roche)), use Ultrasonic disruption.Broken is centrifuged to solution after (15,000g × 30 minutes), by supernatant fraction with 0.8 μM of filtering agent mistake Filter, reclaims filtrate.Filtrate is loaded into ion exchange resin (Hitrap Q HP (1mL)), mixing 20mM PIPES are used (pH6.1,150mM NaCl, buffer solution C) and 20mM PIPES (pH6.1,1000mM NaCl, buffer solution D) solution purification. Each reclaim only makes buffer solution C (NaCl concentration 150mM) flow through ion exchange resin (EL0), use buffer solution C first：Buffer solution D's 3:1 mixed solution (NaCl concentration 363mM) dissolution (EL1), use buffer solution C：The 1 of buffer solution D:1 mixed solution (NaCl concentration 575mM) dissolution (EL2), use buffer solution C：The 1 of buffer solution D:3 mixed solution (NaCl concentration 788mM) dissolutions (EL3), only finally With the fraction of buffer solution D (NaCl concentration 1000mM) dissolution (EL4).
DED-HRV3Csite-NQ01 is detected similarly to Example 1.As a result it is shown in Figure 29.In Figure 29, M is molecular weight Mark, swimming lane 1 are that homogenate solution, swimming lane 2 are that supernatant fraction after centrifugation, swimming lane 3 are pellet fraction, swimming lanes after centrifugation 4~swimming lane 7 be ion exchange resin by fraction 1~4, swimming lane 8 be by buffer solution C (NaCl concentration 150mM) dissolution level It is by buffer solution C to divide (EL0), swimming lane 9：The 3 of buffer solution D:The dissolution fraction (EL1) of 1 mixed solution (NaCl concentration 363mM), Swimming lane 10 is by buffer solution C：The 1 of buffer solution D:The dissolution fraction (EL2) of 1 mixed solution (NaCl concentration 575mM), swimming lane 11 are By buffer solution C：The 1 of buffer solution D:The solution fraction (EL3) of 3 mixed solutions (NaCl concentration 788mM), swimming lane 12 are by buffer solution The electrophoresis result of D (NaCl concentration 1000mM, EL4) dissolution fraction.
【Embodiment 28】
The EL3 of the embodiment 27 of 1/10th capacity is mixed to the EL3 of embodiment 24, in standing at 4 DEG C.After 16 hours, to Reaction solution adds 20mM PIPES (pH6.1) and is diluted to NaCl concentration about 250mM, is loaded into and uses 20mM PIPES On the ion exchange resin (Hitrap Q HP (1mL)) of (pH6.1,150mM NaCl) equilibrating.For that will be reclaimed by fraction (Fr.1-6 (NaCl concentration about 250mM)), the fraction for finally only using buffer solution D (NaCl concentration 1000mM) dissolution (Fr.7-10) Reclaimed respectively.
Target protein EQ01 is detected similarly to Example 1.As a result it is shown in Figure 30.In fig. 30, M is molecular weight markers Thing, swimming lane 1 be the EL3 of embodiment 24, swimming lane 2 be the EL3 of embodiment 27, swimming lane 3 be embodiment 24 EL3 and embodiment 27 EL3 mixed reaction solutions, 4~swimming lane of swimming lane 8 be ion exchange resin by fraction 1~5, swimming lane 9~13 is by buffer solution D The electrophoresis result of dissolution fraction 1~5.
(for the result of embodiment 24~28)
Shown from the result of embodiment 24~28, in the expression-purifying and the purifying of target protein of fused protein In, even if making the pH step-downs of buffer solution, also obtain good separation.
【The explanation of symbol】
10：Ion exchange resin
20：Fused protein
20a：Target protein
20b：Peptide tag
20c：The cut-out site of protein decomposition enzyme
21：Scrambled proteins matter
22：Merge the protein decomposition enzyme of peptide tag
22a：Protein decomposition enzyme
22b：Peptide tag
Sequence table
<110> SYSMEX CORPORATION
<120>Protein purification method and fused protein and its production method containing peptide tag
<130> 15-062CN
<160> 67
<170> PatentIn version 3.5
<210> 1
<211> 12
<212> PRT
<213>Artificial sequence
<220>
<223> DE12
<400> 1
Glu Glu Glu Glu Glu Glu Asp Asp Asp Asp Asp Asp
1 5 10
<210> 2
<211> 18
<212> PRT
<213>Artificial sequence
<220>
<223> DE18
<400> 2
Glu Glu Glu Glu Glu Glu Glu Glu Glu Asp Asp Asp Asp Asp Asp Asp
1 5 10 15
Asp Asp
<210> 3
<211> 24
<212> PRT
<213>Artificial sequence
<220>
<223> DE24
<400> 3
Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Asp Asp Asp Asp
1 5 10 15
Asp Asp Asp Asp Asp Asp Asp Asp
20
<210> 4
<211> 30
<212> PRT
<213>Artificial sequence
<220>
<223> DE30
<400> 4
Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Asp
1 5 10 15
Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp
20 25 30
<210> 5
<211> 36
<212> PRT
<213>Artificial sequence
<220>
<223> DE36
<400> 5
Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu
1 5 10 15
Glu Glu Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp
20 25 30
Asp Asp Asp Asp
35
<210> 6
<211> 88
<212> PRT
<213>Artificial sequence
<220>
<223> DED
<400> 6
Asn Val Glu Gly Lys Thr Gly Asn Ala Thr Asp Glu Glu Glu Glu Glu
1 5 10 15
Glu Glu Glu Glu Glu Glu Glu Asp Asp Asp Asp Asp Asp Asp Asp Asp
20 25 30
Asp Asp Asp Glu Asp Ser Gly Ala Glu Ile Gln Asp Asp Asp Glu Glu
35 40 45
Gly Phe Asp Asp Glu Glu Glu Phe Asp Asp Asp Asp Asp Asp Glu His
50 55 60
Asp Asp Asp Asp Leu Glu Asn Glu Glu Asn Glu Leu Glu Glu Leu Glu
65 70 75 80
Glu Arg Val Glu Ala Arg Lys Lys
85
<210> 7
<211> 44
<212> PRT
<213>Artificial sequence
<220>
<223> DES
<400> 7
Asp Leu Ser Asn Val Glu Gly Lys Thr Gly Asn Ala Thr Asp Glu Glu
1 5 10 15
Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Asp Asp Asp Asp Asp Asp
20 25 30
Asp Asp Asp Asp Asp Asp Glu Asp Ser Gly Ala Glu
35 40
<210> 8
<211> 24
<212> PRT
<213>Artificial sequence
<220>
<223> EO24
<400> 8
Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu
1 5 10 15
Glu Glu Glu Glu Glu Glu Glu Glu
20
<210> 9
<211> 274
<212> PRT
<213>Artificial sequence
<220>
<223> NQ01
<400> 9
Met Val Gly Arg Arg Ala Leu Ile Val Leu Ala His Ser Glu Arg Thr
1 5 10 15
Ser Phe Asn Tyr Ala Met Lys Glu Ala Ala Ala Ala Ala Leu Lys Lys
20 25 30
Lys Gly Trp Glu Val Val Glu Ser Asp Leu Tyr Ala Met Asn Phe Asn
35 40 45
Pro Ile Ile Ser Arg Lys Asp Ile Thr Gly Lys Leu Lys Asp Pro Ala
50 55 60
Asn Phe Gln Tyr Pro Ala Glu Ser Val Leu Ala Tyr Lys Glu Gly His
65 70 75 80
Leu Ser Pro Asp Ile Val Ala Glu Gln Lys Lys Leu Glu Ala Ala Asp
85 90 95
Leu Val Ile Phe Gln Phe Pro Leu Gln Trp Phe Gly Val Pro Ala Ile
100 105 110
Leu Lys Gly Trp Phe Glu Arg Val Phe Ile Gly Glu Phe Ala Tyr Thr
115 120 125
Tyr Ala Ala Met Tyr Asp Lys Gly Pro Phe Arg Ser Lys Lys Ala Val
130 135 140
Leu Ser Ile Thr Thr Gly Gly Ser Gly Ser Met Tyr Ser Leu Gln Gly
145 150 155 160
Ile His Gly Asp Met Asn Val Ile Leu Trp Pro Ile Gln Ser Gly Ile
165 170 175
Leu His Phe Cys Gly Phe Gln Val Leu Glu Pro Gln Leu Thr Tyr Ser
180 185 190
Ile Gly His Thr Pro Ala Asp Ala Arg Ile Gln Ile Leu Glu Gly Trp
195 200 205
Lys Lys Arg Leu Glu Asn Ile Trp Asp Glu Thr Pro Leu Tyr Phe Ala
210 215 220
Pro Ser Ser Leu Phe Asp Leu Asn Phe Gln Ala Gly Phe Leu Met Lys
225 230 235 240
Lys Glu Val Gln Asp Glu Glu Lys Asn Lys Lys Phe Gly Leu Ser Val
245 250 255
Gly His His Leu Gly Lys Ser Ile Pro Thr Asp Asn Gln Ile Lys Ala
260 265 270
Arg Lys
<210> 10
<211> 546
<212> PRT
<213>Artificial sequence
<220>
<223>Luciferase
<400> 10
Glu Asp Ala Lys Asn Ile Lys Lys Gly Pro Ala Pro Phe Tyr Pro Leu
1 5 10 15
Glu Asp Gly Thr Ala Gly Glu Gln Leu His Lys Ala Met Lys Arg Tyr
20 25 30
Ala Leu Val Pro Gly Thr Ile Ala Phe Thr Asp Ala His Ile Glu Val
35 40 45
Asn Ile Thr Tyr Ala Glu Tyr Phe Glu Met Ser Val Arg Leu Ala Glu
50 55 60
Ala Met Lys Arg Tyr Gly Leu Asn Thr Asn His Arg Ile Val Val Cys
65 70 75 80
Ser Glu Asn Ser Leu Gln Phe Phe Met Pro Val Leu Gly Ala Leu Phe
85 90 95
Ile Gly Val Ala Val Ala Pro Ala Asn Asp Ile Tyr Asn Glu Arg Glu
100 105 110
Leu Leu Asn Ser Met Asn Ile Ser Gln Pro Thr Val Val Phe Val Ser
115 120 125
Lys Lys Gly Leu Gln Lys Ile Leu Asn Val Gln Lys Lys Leu Pro Ile
130 135 140
Ile Gln Lys Ile Ile Ile Met Asp Ser Lys Thr Asp Tyr Gln Gly Phe
145 150 155 160
Gln Ser Met Tyr Thr Phe Val Thr Ser His Leu Pro Pro Gly Phe Asn
165 170 175
Glu Tyr Asp Phe Val Pro Glu Ser Phe Asp Arg Asp Lys Thr Ile Ala
180 185 190
Leu Ile Met Asn Ser Ser Gly Ser Thr Gly Leu Pro Lys Gly Val Ala
195 200 205
Leu Pro His Arg Thr Ala Cys Val Arg Phe Ser His Ala Arg Asp Pro
210 215 220
Ile Phe Gly Asn Gln Ile Ile Pro Asp Thr Ala Ile Leu Ser Val Val
225 230 235 240
Pro Phe His His Gly Phe Gly Met Phe Thr Thr Leu Gly Tyr Leu Ile
245 250 255
Cys Gly Phe Arg Val Val Leu Met Tyr Arg Phe Glu Glu Glu Leu Phe
260 265 270
Leu Arg Ser Leu Gln Asp Tyr Lys Ile Gln Ser Ala Leu Leu Val Pro
275 280 285
Thr Leu Phe Ser Phe Phe Ala Lys Ser Thr Leu Ile Asp Lys Tyr Asp
290 295 300
Leu Ser Asn Leu His Glu Ile Ala Ser Gly Gly Ala Pro Leu Ser Lys
305 310 315 320
Glu Val Gly Glu Ala Val Ala Lys Arg Phe His Leu Pro Gly Ile Arg
325 330 335
Gln Gly Tyr Gly Leu Thr Glu Thr Thr Ser Ala Ile Leu Ile Thr Pro
340 345 350
Glu Gly Asp Asp Lys Pro Gly Ala Val Gly Lys Val Val Pro Phe Phe
355 360 365
Glu Ala Lys Val Val Asp Leu Asp Thr Gly Lys Thr Leu Gly Val Asn
370 375 380
Gln Arg Gly Glu Leu Cys Val Arg Gly Pro Met Ile Met Ser Gly Tyr
385 390 395 400
Val Asn Asn Pro Glu Ala Thr Asn Ala Leu Ile Asp Lys Asp Gly Trp
405 410 415
Leu His Ser Gly Asp Ile Ala Tyr Trp Asp Glu Asp Glu His Phe Phe
420 425 430
Ile Val Asp Arg Leu Lys Ser Leu Ile Lys Tyr Lys Gly Tyr Gln Val
435 440 445
Ala Pro Ala Glu Leu Glu Ser Ile Leu Leu Gln His Pro Asn Ile Phe
450 455 460
Asp Ala Gly Val Ala Gly Leu Pro Asp Asp Asp Ala Gly Glu Leu Pro
465 470 475 480
Ala Ala Val Val Val Leu Glu His Gly Lys Thr Met Thr Glu Lys Glu
485 490 495
Ile Val Asp Tyr Val Ala Ser Gln Val Thr Thr Ala Lys Lys Leu Arg
500 505 510
Gly Gly Val Val Phe Val Asp Glu Val Pro Lys Gly Leu Thr Gly Lys
515 520 525
Leu Asp Ala Arg Lys Ile Arg Glu Ile Leu Ile Lys Ala Lys Lys Gly
530 535 540
Gly Lys
545
<210> 11
<211> 202
<212> PRT
<213>Artificial sequence
<220> HRV3C
<400> 11
Asp Leu Val Pro Arg Gly Ser Pro Glu Phe Pro Gly Arg Leu Glu Arg
1 5 10 15
Pro His Arg Asp Gly Pro Asn Thr Glu Phe Ala Leu Ser Leu Leu Arg
20 25 30
Lys Asn Ile Met Thr Ile Thr Thr Ser Lys Gly Glu Phe Thr Gly Leu
35 40 45
Gly Ile His Asp Arg Val Cys Val Ile Pro Thr His Ala Gln Pro Gly
50 55 60
Asp Asp Val Leu Val Asn Gly Gln Lys Ile Arg Val Lys Asp Lys Tyr
65 70 75 80
Lys Leu Val Asp Pro Glu Asn Ile Asn Leu Glu Leu Thr Val Leu Thr
85 90 95
Leu Asp Arg Asn Glu Lys Phe Arg Asp Ile Arg Gly Phe Ile Ser Glu
100 105 110
Asp Leu Glu Gly Val Asp Ala Thr Leu Val Val His Ser Asn Asn Phe
115 120 125
Thr Asn Thr Ile Leu Glu Val Gly Pro Val Thr Met Ala Gly Leu Ile
130 135 140
Asn Leu Ser Ser Thr Pro Thr Asn Arg Met Ile Arg Tyr Asp Tyr Ala
145 150 155 160
Thr Lys Thr Gly Gln Cys Gly Gly Val Leu Cys Ala Thr Gly Lys Ile
165 170 175
Phe Gly Ile His Val Gly Gly Asn Gly Arg Gln Gly Phe Ser Ala Gln
180 185 190
Leu Lys Lys Gln Tyr Phe Val Glu Lys Gln
195 200
<210> 12
<211> 298
<212> PRT
<213>Artificial sequence
<220>
<223> DE12-HRV3Csite-NQO1
<400> 12
Met Glu Glu Glu Glu Glu Glu Asp Asp Asp Asp Asp Asp Lys Gly Thr
1 5 10 15
Leu Glu Val Leu Phe Gln Gly Pro Met Val Gly Arg Arg Ala Leu Ile
20 25 30
Val Leu Ala His Ser Glu Arg Thr Ser Phe Asn Tyr Ala Met Lys Glu
35 40 45
Ala Ala Ala Ala Ala Leu Lys Lys Lys Gly Trp Glu Val Val Glu Ser
50 55 60
Asp Leu Tyr Ala Met Asn Phe Asn Pro Ile Ile Ser Arg Lys Asp Ile
65 70 75 80
Thr Gly Lys Leu Lys Asp Pro Ala Asn Phe Gln Tyr Pro Ala Glu Ser
85 90 95
Val Leu Ala Tyr Lys Glu Gly His Leu Ser Pro Asp Ile Val Ala Glu
100 105 110
Gln Lys Lys Leu Glu Ala Ala Asp Leu Val Ile Phe Gln Phe Pro Leu
115 120 125
Gln Trp Phe Gly Val Pro Ala Ile Leu Lys Gly Trp Phe Glu Arg Val
130 135 140
Phe Ile Gly Glu Phe Ala Tyr Thr Tyr Ala Ala Met Tyr Asp Lys Gly
145 150 155 160
Pro Phe Arg Ser Lys Lys Ala Val Leu Ser Ile Thr Thr Gly Gly Ser
165 170 175
Gly Ser Met Tyr Ser Leu Gln Gly Ile His Gly Asp Met Asn Val Ile
180 185 190
Leu Trp Pro Ile Gln Ser Gly Ile Leu His Phe Cys Gly Phe Gln Val
195 200 205
Leu Glu Pro Gln Leu Thr Tyr Ser Ile Gly His Thr Pro Ala Asp Ala
210 215 220
Arg Ile Gln Ile Leu Glu Gly Trp Lys Lys Arg Leu Glu Asn Ile Trp
225 230 235 240
Asp Glu Thr Pro Leu Tyr Phe Ala Pro Ser Ser Leu Phe Asp Leu Asn
245 250 255
Phe Gln Ala Gly Phe Leu Met Lys Lys Glu Val Gln Asp Glu Glu Lys
260 265 270
Asn Lys Lys Phe Gly Leu Ser Val Gly His His Leu Gly Lys Ser Ile
275 280 285
Pro Thr Asp Asn Gln Ile Lys Ala Arg Lys
290 295
<210> 13
<211> 304
<212> PRT
<213>Artificial sequence
<220>
<223> DE18-HRV3Csite-NQO1
<400> 13
Met Glu Glu Glu Glu Glu Glu Glu Glu Glu Asp Asp Asp Asp Asp Asp
1 5 10 15
Asp Asp Asp Lys Gly Thr Leu Glu Val Leu Phe Gln Gly Pro Met Val
20 25 30
Gly Arg Arg Ala Leu Ile Val Leu Ala His Ser Glu Arg Thr Ser Phe
35 40 45
Asn Tyr Ala Met Lys Glu Ala Ala Ala Ala Ala Leu Lys Lys Lys Gly
50 55 60
Trp Glu Val Val Glu Ser Asp Leu Tyr Ala Met Asn Phe Asn Pro Ile
65 70 75 80
Ile Ser Arg Lys Asp Ile Thr Gly Lys Leu Lys Asp Pro Ala Asn Phe
85 90 95
Gln Tyr Pro Ala Glu Ser Val Leu Ala Tyr Lys Glu Gly His Leu Ser
100 105 110
Pro Asp Ile Val Ala Glu Gln Lys Lys Leu Glu Ala Ala Asp Leu Val
115 120 125
Ile Phe Gln Phe Pro Leu Gln Trp Phe Gly Val Pro Ala Ile Leu Lys
130 135 140
Gly Trp Phe Glu Arg Val Phe Ile Gly Glu Phe Ala Tyr Thr Tyr Ala
145 150 155 160
Ala Met Tyr Asp Lys Gly Pro Phe Arg Ser Lys Lys Ala Val Leu Ser
165 170 175
Ile Thr Thr Gly Gly Ser Gly Ser Met Tyr Ser Leu Gln Gly Ile His
180 185 190
Gly Asp Met Asn Val Ile Leu Trp Pro Ile Gln Ser Gly Ile Leu His
195 200 205
Phe Cys Gly Phe Gln Val Leu Glu Pro Gln Leu Thr Tyr Ser Ile Gly
210 215 220
His Thr Pro Ala Asp Ala Arg Ile Gln Ile Leu Glu Gly Trp Lys Lys
225 230 235 240
Arg Leu Glu Asn Ile Trp Asp Glu Thr Pro Leu Tyr Phe Ala Pro Ser
245 250 255
Ser Leu Phe Asp Leu Asn Phe Gln Ala Gly Phe Leu Met Lys Lys Glu
260 265 270
Val Gln Asp Glu Glu Lys Asn Lys Lys Phe Gly Leu Ser Val Gly His
275 280 285
His Leu Gly Lys Ser Ile Pro Thr Asp Asn Gln Ile Lys Ala Arg Lys
290 295 300
<210> 14
<211> 310
<212> PRT
<213>Artificial sequence
<220>
<223> DE24-HRV3Csite-NQO1
<400> 14
Met Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Asp Asp Asp
1 5 10 15
Asp Asp Asp Asp Asp Asp Asp Asp Asp Lys Gly Thr Leu Glu Val Leu
20 25 30
Phe Gln Gly Pro Met Val Gly Arg Arg Ala Leu Ile Val Leu Ala His
35 40 45
Ser Glu Arg Thr Ser Phe Asn Tyr Ala Met Lys Glu Ala Ala Ala Ala
50 55 60
Ala Leu Lys Lys Lys Gly Trp Glu Val Val Glu Ser Asp Leu Tyr Ala
65 70 75 80
Met Asn Phe Asn Pro Ile Ile Ser Arg Lys Asp Ile Thr Gly Lys Leu
85 90 95
Lys Asp Pro Ala Asn Phe Gln Tyr Pro Ala Glu Ser Val Leu Ala Tyr
100 105 110
Lys Glu Gly His Leu Ser Pro Asp Ile Val Ala Glu Gln Lys Lys Leu
115 120 125
Glu Ala Ala Asp Leu Val Ile Phe Gln Phe Pro Leu Gln Trp Phe Gly
130 135 140
Val Pro Ala Ile Leu Lys Gly Trp Phe Glu Arg Val Phe Ile Gly Glu
145 150 155 160
Phe Ala Tyr Thr Tyr Ala Ala Met Tyr Asp Lys Gly Pro Phe Arg Ser
165 170 175
Lys Lys Ala Val Leu Ser Ile Thr Thr Gly Gly Ser Gly Ser Met Tyr
180 185 190
Ser Leu Gln Gly Ile His Gly Asp Met Asn Val Ile Leu Trp Pro Ile
195 200 205
Gln Ser Gly Ile Leu His Phe Cys Gly Phe Gln Val Leu Glu Pro Gln
210 215 220
Leu Thr Tyr Ser Ile Gly His Thr Pro Ala Asp Ala Arg Ile Gln Ile
225 230 235 240
Leu Glu Gly Trp Lys Lys Arg Leu Glu Asn Ile Trp Asp Glu Thr Pro
245 250 255
Leu Tyr Phe Ala Pro Ser Ser Leu Phe Asp Leu Asn Phe Gln Ala Gly
260 265 270
Phe Leu Met Lys Lys Glu Val Gln Asp Glu Glu Lys Asn Lys Lys Phe
275 280 285
Gly Leu Ser Val Gly His His Leu Gly Lys Ser Ile Pro Thr Asp Asn
290 295 300
Gln Ile Lys Ala Arg Lys
305 310
<210> 15
<211> 316
<212> PRT
<213>Artificial sequence
<220>
<223> DE30-HRV3Csite-NQO1
<400> 15
Met Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu
1 5 10 15
Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Lys
20 25 30
Gly Thr Leu Glu Val Leu Phe Gln Gly Pro Met Val Gly Arg Arg Ala
35 40 45
Leu Ile Val Leu Ala His Ser Glu Arg Thr Ser Phe Asn Tyr Ala Met
50 55 60
Lys Glu Ala Ala Ala Ala Ala Leu Lys Lys Lys Gly Trp Glu Val Val
65 70 75 80
Glu Ser Asp Leu Tyr Ala Met Asn Phe Asn Pro Ile Ile Ser Arg Lys
85 90 95
Asp Ile Thr Gly Lys Leu Lys Asp Pro Ala Asn Phe Gln Tyr Pro Ala
100 105 110
Glu Ser Val Leu Ala Tyr Lys Glu Gly His Leu Ser Pro Asp Ile Val
115 120 125
Ala Glu Gln Lys Lys Leu Glu Ala Ala Asp Leu Val Ile Phe Gln Phe
130 135 140
Pro Leu Gln Trp Phe Gly Val Pro Ala Ile Leu Lys Gly Trp Phe Glu
145 150 155 160
Arg Val Phe Ile Gly Glu Phe Ala Tyr Thr Tyr Ala Ala Met Tyr Asp
165 170 175
Lys Gly Pro Phe Arg Ser Lys Lys Ala Val Leu Ser Ile Thr Thr Gly
180 185 190
Gly Ser Gly Ser Met Tyr Ser Leu Gln Gly Ile His Gly Asp Met Asn
195 200 205
Val Ile Leu Trp Pro Ile Gln Ser Gly Ile Leu His Phe Cys Gly Phe
210 215 220
Gln Val Leu Glu Pro Gln Leu Thr Tyr Ser Ile Gly His Thr Pro Ala
225 230 235 240
Asp Ala Arg Ile Gln Ile Leu Glu Gly Trp Lys Lys Arg Leu Glu Asn
245 250 255
Ile Trp Asp Glu Thr Pro Leu Tyr Phe Ala Pro Ser Ser Leu Phe Asp
260 265 270
Leu Asn Phe Gln Ala Gly Phe Leu Met Lys Lys Glu Val Gln Asp Glu
275 280 285
Glu Lys Asn Lys Lys Phe Gly Leu Ser Val Gly His His Leu Gly Lys
290 295 300
Ser Ile Pro Thr Asp Asn Gln Ile Lys Ala Arg Lys
305 310 315
<210> 16
<211> 322
<212> PRT
<213>Artificial sequence
<220>
<223> DE36-HRV3Csite-NQO1
<400> 16
Met Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu
1 5 10 15
Glu Glu Glu Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp
20 25 30
Asp Asp Asp Asp Asp Lys Gly Thr Leu Glu Val Leu Phe Gln Gly Pro
35 40 45
Met Val Gly Arg Arg Ala Leu Ile Val Leu Ala His Ser Glu Arg Thr
50 55 60
Ser Phe Asn Tyr Ala Met Lys Glu Ala Ala Ala Ala Ala Leu Lys Lys
65 70 75 80
Lys Gly Trp Glu Val Val Glu Ser Asp Leu Tyr Ala Met Asn Phe Asn
85 90 95
Pro Ile Ile Ser Arg Lys Asp Ile Thr Gly Lys Leu Lys Asp Pro Ala
100 105 110
Asn Phe Gln Tyr Pro Ala Glu Ser Val Leu Ala Tyr Lys Glu Gly His
115 120 125
Leu Ser Pro Asp Ile Val Ala Glu Gln Lys Lys Leu Glu Ala Ala Asp
130 135 140
Leu Val Ile Phe Gln Phe Pro Leu Gln Trp Phe Gly Val Pro Ala Ile
145 150 155 160
Leu Lys Gly Trp Phe Glu Arg Val Phe Ile Gly Glu Phe Ala Tyr Thr
165 170 175
Tyr Ala Ala Met Tyr Asp Lys Gly Pro Phe Arg Ser Lys Lys Ala Val
180 185 190
Leu Ser Ile Thr Thr Gly Gly Ser Gly Ser Met Tyr Ser Leu Gln Gly
195 200 205
Ile His Gly Asp Met Asn Val Ile Leu Trp Pro Ile Gln Ser Gly Ile
210 215 220
Leu His Phe Cys Gly Phe Gln Val Leu Glu Pro Gln Leu Thr Tyr Ser
225 230 235 240
Ile Gly His Thr Pro Ala Asp Ala Arg Ile Gln Ile Leu Glu Gly Trp
245 250 255
Lys Lys Arg Leu Glu Asn Ile Trp Asp Glu Thr Pro Leu Tyr Phe Ala
260 265 270
Pro Ser Ser Leu Phe Asp Leu Asn Phe Gln Ala Gly Phe Leu Met Lys
275 280 285
Lys Glu Val Gln Asp Glu Glu Lys Asn Lys Lys Phe Gly Leu Ser Val
290 295 300
Gly His His Leu Gly Lys Ser Ile Pro Thr Asp Asn Gln Ile Lys Ala
305 310 315 320
Arg Lys
<210> 17
<211> 388
<212> PRT
<213>Artificial sequence
<220>
<223> DED-HRV3Csite-NQO1
<400> 17
Met Asp Tyr Lys Asp Asp Asp Asp Lys Gly Met Ala Ser Asp Leu Ser
1 5 10 15
Asn Val Glu Gly Lys Thr Gly Asn Ala Thr Asp Glu Glu Glu Glu Glu
20 25 30
Glu Glu Glu Glu Glu Glu Glu Asp Asp Asp Asp Asp Asp Asp Asp Asp
35 40 45
Asp Asp Asp Glu Asp Ser Gly Ala Glu Ile Gln Asp Asp Asp Glu Glu
50 55 60
Gly Phe Asp Asp Glu Glu Glu Phe Asp Asp Asp Asp Asp Asp Glu His
65 70 75 80
Asp Asp Asp Asp Leu Glu Asn Glu Glu Asn Glu Leu Glu Glu Leu Glu
85 90 95
Glu Arg Val Glu Ala Arg Lys Lys Ala Ser Leu Glu Val Leu Phe Gln
100 105 110
Gly Pro Met Val Gly Arg Arg Ala Leu Ile Val Leu Ala His Ser Glu
115 120 125
Arg Thr Ser Phe Asn Tyr Ala Met Lys Glu Ala Ala Ala Ala Ala Leu
130 135 140
Lys Lys Lys Gly Trp Glu Val Val Glu Ser Asp Leu Tyr Ala Met Asn
145 150 155 160
Phe Asn Pro Ile Ile Ser Arg Lys Asp Ile Thr Gly Lys Leu Lys Asp
165 170 175
Pro Ala Asn Phe Gln Tyr Pro Ala Glu Ser Val Leu Ala Tyr Lys Glu
180 185 190
Gly His Leu Ser Pro Asp Ile Val Ala Glu Gln Lys Lys Leu Glu Ala
195 200 205
Ala Asp Leu Val Ile Phe Gln Phe Pro Leu Gln Trp Phe Gly Val Pro
210 215 220
Ala Ile Leu Lys Gly Trp Phe Glu Arg Val Phe Ile Gly Glu Phe Ala
225 230 235 240
Tyr Thr Tyr Ala Ala Met Tyr Asp Lys Gly Pro Phe Arg Ser Lys Lys
245 250 255
Ala Val Leu Ser Ile Thr Thr Gly Gly Ser Gly Ser Met Tyr Ser Leu
260 265 270
Gln Gly Ile His Gly Asp Met Asn Val Ile Leu Trp Pro Ile Gln Ser
275 280 285
Gly Ile Leu His Phe Cys Gly Phe Gln Val Leu Glu Pro Gln Leu Thr
290 295 300
Tyr Ser Ile Gly His Thr Pro Ala Asp Ala Arg Ile Gln Ile Leu Glu
305 310 315 320
Gly Trp Lys Lys Arg Leu Glu Asn Ile Trp Asp Glu Thr Pro Leu Tyr
325 330 335
Phe Ala Pro Ser Ser Leu Phe Asp Leu Asn Phe Gln Ala Gly Phe Leu
340 345 350
Met Lys Lys Glu Val Gln Asp Glu Glu Lys Asn Lys Lys Phe Gly Leu
355 360 365
Ser Val Gly His His Leu Gly Lys Ser Ile Pro Thr Asp Asn Gln Ile
370 375 380
Lys Ala Arg Lys
385
<210> 18
<211> 341
<212> PRT
<213>Artificial sequence
<220>
<223> DES-HRV3Csite-NQO1
<400> 18
Met Asp Tyr Lys Asp Asp Asp Asp Lys Gly Met Ala Ser Asp Leu Ser
1 5 10 15
Asn Val Glu Gly Lys Thr Gly Asn Ala Thr Asp Glu Glu Glu Glu Glu
20 25 30
Glu Glu Glu Glu Glu Glu Glu Asp Asp Asp Asp Asp Asp Asp Asp Asp
35 40 45
Asp Asp Asp Glu Asp Ser Gly Ala Glu Ala Ser Leu Glu Val Leu Phe
50 55 60
Gln Gly Pro Met Val Gly Arg Arg Ala Leu Ile Val Leu Ala His Ser
65 70 75 80
Glu Arg Thr Ser Phe Asn Tyr Ala Met Lys Glu Ala Ala Ala Ala Ala
85 90 95
Leu Lys Lys Lys Gly Trp Glu Val Val Glu Ser Asp Leu Tyr Ala Met
100 105 110
Asn Phe Asn Pro Ile Ile Ser Arg Lys Asp Ile Thr Gly Lys Leu Lys
115 120 125
Asp Pro Ala Asn Phe Gln Tyr Pro Ala Glu Ser Val Leu Ala Tyr Lys
130 135 140
Glu Gly His Leu Ser Pro Asp Ile Val Ala Glu Gln Lys Lys Leu Glu
145 150 155 160
Ala Ala Asp Leu Val Ile Phe Gln Phe Pro Leu Gln Trp Phe Gly Val
165 170 175
Pro Ala Ile Leu Lys Gly Trp Phe Glu Arg Val Phe Ile Gly Glu Phe
180 185 190
Ala Tyr Thr Tyr Ala Ala Met Tyr Asp Lys Gly Pro Phe Arg Ser Lys
195 200 205
Lys Ala Val Leu Ser Ile Thr Thr Gly Gly Ser Gly Ser Met Tyr Ser
210 215 220
Leu Gln Gly Ile His Gly Asp Met Asn Val Ile Leu Trp Pro Ile Gln
225 230 235 240
Ser Gly Ile Leu His Phe Cys Gly Phe Gln Val Leu Glu Pro Gln Leu
245 250 255
Thr Tyr Ser Ile Gly His Thr Pro Ala Asp Ala Arg Ile Gln Ile Leu
260 265 270
Glu Gly Trp Lys Lys Arg Leu Glu Asn Ile Trp Asp Glu Thr Pro Leu
275 280 285
Tyr Phe Ala Pro Ser Ser Leu Phe Asp Leu Asn Phe Gln Ala Gly Phe
290 295 300
Leu Met Lys Lys Glu Val Gln Asp Glu Glu Lys Asn Lys Lys Phe Gly
305 310 315 320
Leu Ser Val Gly His His Leu Gly Lys Ser Ile Pro Thr Asp Asn Gln
325 330 335
Ile Lys Ala Arg Lys
340
<210> 19
<211> 321
<212> PRT
<213>Artificial sequence
<220>
<223> EO24-HRV3Csite-NQO1
<400> 19
Met Asp Tyr Lys Asp Asp Asp Asp Lys Gly Met Ala Ser Glu Glu Glu
1 5 10 15
Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu
20 25 30
Glu Glu Glu Glu Glu Ala Ser Leu Glu Val Leu Phe Gln Gly Pro Met
35 40 45
Val Gly Arg Arg Ala Leu Ile Val Leu Ala His Ser Glu Arg Thr Ser
50 55 60
Phe Asn Tyr Ala Met Lys Glu Ala Ala Ala Ala Ala Leu Lys Lys Lys
65 70 75 80
Gly Trp Glu Val Val Glu Ser Asp Leu Tyr Ala Met Asn Phe Asn Pro
85 90 95
Ile Ile Ser Arg Lys Asp Ile Thr Gly Lys Leu Lys Asp Pro Ala Asn
100 105 110
Phe Gln Tyr Pro Ala Glu Ser Val Leu Ala Tyr Lys Glu Gly His Leu
115 120 125
Ser Pro Asp Ile Val Ala Glu Gln Lys Lys Leu Glu Ala Ala Asp Leu
130 135 140
Val Ile Phe Gln Phe Pro Leu Gln Trp Phe Gly Val Pro Ala Ile Leu
145 150 155 160
Lys Gly Trp Phe Glu Arg Val Phe Ile Gly Glu Phe Ala Tyr Thr Tyr
165 170 175
Ala Ala Met Tyr Asp Lys Gly Pro Phe Arg Ser Lys Lys Ala Val Leu
180 185 190
Ser Ile Thr Thr Gly Gly Ser Gly Ser Met Tyr Ser Leu Gln Gly Ile
195 200 205
His Gly Asp Met Asn Val Ile Leu Trp Pro Ile Gln Ser Gly Ile Leu
210 215 220
His Phe Cys Gly Phe Gln Val Leu Glu Pro Gln Leu Thr Tyr Ser Ile
225 230 235 240
Gly His Thr Pro Ala Asp Ala Arg Ile Gln Ile Leu Glu Gly Trp Lys
245 250 255
Lys Arg Leu Glu Asn Ile Trp Asp Glu Thr Pro Leu Tyr Phe Ala Pro
260 265 270
Ser Ser Leu Phe Asp Leu Asn Phe Gln Ala Gly Phe Leu Met Lys Lys
275 280 285
Glu Val Gln Asp Glu Glu Lys Asn Lys Lys Phe Gly Leu Ser Val Gly
290 295 300
His His Leu Gly Lys Ser Ile Pro Thr Asp Asn Gln Ile Lys Ala Arg
305 310 315 320
Lys
<210> 20
<211> 386
<212> PRT
<213>Artificial sequence
<220>
<223> NQO1-HRV3Csite-DED
<400> 20
Met Val Gly Arg Arg Ala Leu Ile Val Leu Ala His Ser Glu Arg Thr
1 5 10 15
Ser Phe Asn Tyr Ala Met Lys Glu Ala Ala Ala Ala Ala Leu Lys Lys
20 25 30
Lys Gly Trp Glu Val Val Glu Ser Asp Leu Tyr Ala Met Asn Phe Asn
35 40 45
Pro Ile Ile Ser Arg Lys Asp Ile Thr Gly Lys Leu Lys Asp Pro Ala
50 55 60
Asn Phe Gln Tyr Pro Ala Glu Ser Val Leu Ala Tyr Lys Glu Gly His
65 70 75 80
Leu Ser Pro Asp Ile Val Ala Glu Gln Lys Lys Leu Glu Ala Ala Asp
85 90 95
Leu Val Ile Phe Gln Phe Pro Leu Gln Trp Phe Gly Val Pro Ala Ile
100 105 110
Leu Lys Gly Trp Phe Glu Arg Val Phe Ile Gly Glu Phe Ala Tyr Thr
115 120 125
Tyr Ala Ala Met Tyr Asp Lys Gly Pro Phe Arg Ser Lys Lys Ala Val
130 135 140
Leu Ser Ile Thr Thr Gly Gly Ser Gly Ser Met Tyr Ser Leu Gln Gly
145 150 155 160
Ile His Gly Asp Met Asn Val Ile Leu Trp Pro Ile Gln Ser Gly Ile
165 170 175
Leu His Phe Cys Gly Phe Gln Val Leu Glu Pro Gln Leu Thr Tyr Ser
180 185 190
Ile Gly His Thr Pro Ala Asp Ala Arg Ile Gln Ile Leu Glu Gly Trp
195 200 205
Lys Lys Arg Leu Glu Asn Ile Trp Asp Glu Thr Pro Leu Tyr Phe Ala
210 215 220
Pro Ser Ser Leu Phe Asp Leu Asn Phe Gln Ala Gly Phe Leu Met Lys
225 230 235 240
Lys Glu Val Gln Asp Glu Glu Lys Asn Lys Lys Phe Gly Leu Ser Val
245 250 255
Gly His His Leu Gly Lys Ser Ile Pro Thr Asp Asn Gln Ile Lys Ala
260 265 270
Arg Lys Gly Leu Glu Val Leu Phe Gln Gly Pro Ala Ser Asp Leu Ser
275 280 285
Asn Val Glu Gly Lys Thr Gly Asn Ala Thr Asp Glu Glu Glu Glu Glu
290 295 300
Glu Glu Glu Glu Glu Glu Glu Asp Asp Asp Asp Asp Asp Asp Asp Asp
305 310 315 320
Asp Asp Asp Glu Asp Ser Gly Ala Glu Ile Gln Asp Asp Asp Glu Glu
325 330 335
Gly Phe Asp Asp Glu Glu Glu Phe Asp Asp Asp Asp Asp Asp Glu His
340 345 350
Asp Asp Asp Asp Leu Glu Asn Glu Glu Asn Glu Leu Glu Glu Leu Glu
355 360 365
Glu Arg Val Glu Ala Arg Lys Lys Ala Ser Asp Tyr Lys Asp Asp Asp
370 375 380
Asp Lys
385
<210> 21
<211> 339
<212> PRT
<213>Artificial sequence
<220>
<223> NQO1-HRV3Csite-DES
<400> 21
Met Val Gly Arg Arg Ala Leu Ile Val Leu Ala His Ser Glu Arg Thr
1 5 10 15
Ser Phe Asn Tyr Ala Met Lys Glu Ala Ala Ala Ala Ala Leu Lys Lys
20 25 30
Lys Gly Trp Glu Val Val Glu Ser Asp Leu Tyr Ala Met Asn Phe Asn
35 40 45
Pro Ile Ile Ser Arg Lys Asp Ile Thr Gly Lys Leu Lys Asp Pro Ala
50 55 60
Asn Phe Gln Tyr Pro Ala Glu Ser Val Leu Ala Tyr Lys Glu Gly His
65 70 75 80
Leu Ser Pro Asp Ile Val Ala Glu Gln Lys Lys Leu Glu Ala Ala Asp
85 90 95
Leu Val Ile Phe Gln Phe Pro Leu Gln Trp Phe Gly Val Pro Ala Ile
100 105 110
Leu Lys Gly Trp Phe Glu Arg Val Phe Ile Gly Glu Phe Ala Tyr Thr
115 120 125
Tyr Ala Ala Met Tyr Asp Lys Gly Pro Phe Arg Ser Lys Lys Ala Val
130 135 140
Leu Ser Ile Thr Thr Gly Gly Ser Gly Ser Met Tyr Ser Leu Gln Gly
145 150 155 160
Ile His Gly Asp Met Asn Val Ile Leu Trp Pro Ile Gln Ser Gly Ile
165 170 175
Leu His Phe Cys Gly Phe Gln Val Leu Glu Pro Gln Leu Thr Tyr Ser
180 185 190
Ile Gly His Thr Pro Ala Asp Ala Arg Ile Gln Ile Leu Glu Gly Trp
195 200 205
Lys Lys Arg Leu Glu Asn Ile Trp Asp Glu Thr Pro Leu Tyr Phe Ala
210 215 220
Pro Ser Ser Leu Phe Asp Leu Asn Phe Gln Ala Gly Phe Leu Met Lys
225 230 235 240
Lys Glu Val Gln Asp Glu Glu Lys Asn Lys Lys Phe Gly Leu Ser Val
245 250 255
Gly His His Leu Gly Lys Ser Ile Pro Thr Asp Asn Gln Ile Lys Ala
260 265 270
Arg Lys Gly Leu Glu Val Leu Phe Gln Gly Pro Ala Ser Asp Leu Ser
275 280 285
Asn Val Glu Gly Lys Thr Gly Asn Ala Thr Asp Glu Glu Glu Glu Glu
290 295 300
Glu Glu Glu Glu Glu Glu Glu Asp Asp Asp Asp Asp Asp Asp Asp Asp
305 310 315 320
Asp Asp Asp Glu Asp Ser Gly Ala Glu Ala Ser Asp Tyr Lys Asp Asp
325 330 335
Asp Asp Lys
<210> 22
<211> 660
<212> PRT
<213>Artificial sequence
<220>
<223>DED-HRV3Csite- luciferases
<400> 22
Met Asp Tyr Lys Asp Asp Asp Asp Lys Gly Met Ala Ser Asp Leu Ser
1 5 10 15
Asn Val Glu Gly Lys Thr Gly Asn Ala Thr Asp Glu Glu Glu Glu Glu
20 25 30
Glu Glu Glu Glu Glu Glu Glu Asp Asp Asp Asp Asp Asp Asp Asp Asp
35 40 45
Asp Asp Asp Glu Asp Ser Gly Ala Glu Ile Gln Asp Asp Asp Glu Glu
50 55 60
Gly Phe Asp Asp Glu Glu Glu Phe Asp Asp Asp Asp Asp Asp Glu His
65 70 75 80
Asp Asp Asp Asp Leu Glu Asn Glu Glu Asn Glu Leu Glu Glu Leu Glu
85 90 95
Glu Arg Val Glu Ala Arg Lys Lys Ala Ser Leu Glu Val Leu Phe Gln
100 105 110
Gly Pro Glu Asp Ala Lys Asn Ile Lys Lys Gly Pro Ala Pro Phe Tyr
115 120 125
Pro Leu Glu Asp Gly Thr Ala Gly Glu Gln Leu His Lys Ala Met Lys
130 135 140
Arg Tyr Ala Leu Val Pro Gly Thr Ile Ala Phe Thr Asp Ala His Ile
145 150 155 160
Glu Val Asn Ile Thr Tyr Ala Glu Tyr Phe Glu Met Ser Val Arg Leu
165 170 175
Ala Glu Ala Met Lys Arg Tyr Gly Leu Asn Thr Asn His Arg Ile Val
180 185 190
Val Cys Ser Glu Asn Ser Leu Gln Phe Phe Met Pro Val Leu Gly Ala
195 200 205
Leu Phe Ile Gly Val Ala Val Ala Pro Ala Asn Asp Ile Tyr Asn Glu
210 215 220
Arg Glu Leu Leu Asn Ser Met Asn Ile Ser Gln Pro Thr Val Val Phe
225 230 235 240
Val Ser Lys Lys Gly Leu Gln Lys Ile Leu Asn Val Gln Lys Lys Leu
245 250 255
Pro Ile Ile Gln Lys Ile Ile Ile Met Asp Ser Lys Thr Asp Tyr Gln
260 265 270
Gly Phe Gln Ser Met Tyr Thr Phe Val Thr Ser His Leu Pro Pro Gly
275 280 285
Phe Asn Glu Tyr Asp Phe Val Pro Glu Ser Phe Asp Arg Asp Lys Thr
290 295 300
Ile Ala Leu Ile Met Asn Ser Ser Gly Ser Thr Gly Leu Pro Lys Gly
305 310 315 320
Val Ala Leu Pro His Arg Thr Ala Cys Val Arg Phe Ser His Ala Arg
325 330 335
Asp Pro Ile Phe Gly Asn Gln Ile Ile Pro Asp Thr Ala Ile Leu Ser
340 345 350
Val Val Pro Phe His His Gly Phe Gly Met Phe Thr Thr Leu Gly Tyr
355 360 365
Leu Ile Cys Gly Phe Arg Val Val Leu Met Tyr Arg Phe Glu Glu Glu
370 375 380
Leu Phe Leu Arg Ser Leu Gln Asp Tyr Lys Ile Gln Ser Ala Leu Leu
385 390 395 400
Val Pro Thr Leu Phe Ser Phe Phe Ala Lys Ser Thr Leu Ile Asp Lys
405 410 415
Tyr Asp Leu Ser Asn Leu His Glu Ile Ala Ser Gly Gly Ala Pro Leu
420 425 430
Ser Lys Glu Val Gly Glu Ala Val Ala Lys Arg Phe His Leu Pro Gly
435 440 445
Ile Arg Gln Gly Tyr Gly Leu Thr Glu Thr Thr Ser Ala Ile Leu Ile
450 455 460
Thr Pro Glu Gly Asp Asp Lys Pro Gly Ala Val Gly Lys Val Val Pro
465 470 475 480
Phe Phe Glu Ala Lys Val Val Asp Leu Asp Thr Gly Lys Thr Leu Gly
485 490 495
Val Asn Gln Arg Gly Glu Leu Cys Val Arg Gly Pro Met Ile Met Ser
500 505 510
Gly Tyr Val Asn Asn Pro Glu Ala Thr Asn Ala Leu Ile Asp Lys Asp
515 520 525
Gly Trp Leu His Ser Gly Asp Ile Ala Tyr Trp Asp Glu Asp Glu His
530 535 540
Phe Phe Ile Val Asp Arg Leu Lys Ser Leu Ile Lys Tyr Lys Gly Tyr
545 550 555 560
Gln Val Ala Pro Ala Glu Leu Glu Ser Ile Leu Leu Gln His Pro Asn
565 570 575
Ile Phe Asp Ala Gly Val Ala Gly Leu Pro Asp Asp Asp Ala Gly Glu
580 585 590
Leu Pro Ala Ala Val Val Val Leu Glu His Gly Lys Thr Met Thr Glu
595 600 605
Lys Glu Ile Val Asp Tyr Val Ala Ser Gln Val Thr Thr Ala Lys Lys
610 615 620
Leu Arg Gly Gly Val Val Phe Val Asp Glu Val Pro Lys Gly Leu Thr
625 630 635 640
Gly Lys Leu Asp Ala Arg Lys Ile Arg Glu Ile Leu Ile Lys Ala Lys
645 650 655
Lys Gly Gly Lys
660
<210> 23
<211> 613
<212> PRT
<213>Artificial sequence
<220>
<223>DES-HRV3Csite- luciferases
<400> 23
Met Asp Tyr Lys Asp Asp Asp Asp Lys Gly Met Ala Ser Asp Leu Ser
1 5 10 15
Asn Val Glu Gly Lys Thr Gly Asn Ala Thr Asp Glu Glu Glu Glu Glu
20 25 30
Glu Glu Glu Glu Glu Glu Glu Asp Asp Asp Asp Asp Asp Asp Asp Asp
35 40 45
Asp Asp Asp Glu Asp Ser Gly Ala Glu Ala Ser Leu Glu Val Leu Phe
50 55 60
Gln Gly Pro Glu Asp Ala Lys Asn Ile Lys Lys Gly Pro Ala Pro Phe
65 70 75 80
Tyr Pro Leu Glu Asp Gly Thr Ala Gly Glu Gln Leu His Lys Ala Met
85 90 95
Lys Arg Tyr Ala Leu Val Pro Gly Thr Ile Ala Phe Thr Asp Ala His
100 105 110
Ile Glu Val Asn Ile Thr Tyr Ala Glu Tyr Phe Glu Met Ser Val Arg
115 120 125
Leu Ala Glu Ala Met Lys Arg Tyr Gly Leu Asn Thr Asn His Arg Ile
130 135 140
Val Val Cys Ser Glu Asn Ser Leu Gln Phe Phe Met Pro Val Leu Gly
145 150 155 160
Ala Leu Phe Ile Gly Val Ala Val Ala Pro Ala Asn Asp Ile Tyr Asn
165 170 175
Glu Arg Glu Leu Leu Asn Ser Met Asn Ile Ser Gln Pro Thr Val Val
180 185 190
Phe Val Ser Lys Lys Gly Leu Gln Lys Ile Leu Asn Val Gln Lys Lys
195 200 205
Leu Pro Ile Ile Gln Lys Ile Ile Ile Met Asp Ser Lys Thr Asp Tyr
210 215 220
Gln Gly Phe Gln Ser Met Tyr Thr Phe Val Thr Ser His Leu Pro Pro
225 230 235 240
Gly Phe Asn Glu Tyr Asp Phe Val Pro Glu Ser Phe Asp Arg Asp Lys
245 250 255
Thr Ile Ala Leu Ile Met Asn Ser Ser Gly Ser Thr Gly Leu Pro Lys
260 265 270
Gly Val Ala Leu Pro His Arg Thr Ala Cys Val Arg Phe Ser His Ala
275 280 285
Arg Asp Pro Ile Phe Gly Asn Gln Ile Ile Pro Asp Thr Ala Ile Leu
290 295 300
Ser Val Val Pro Phe His His Gly Phe Gly Met Phe Thr Thr Leu Gly
305 310 315 320
Tyr Leu Ile Cys Gly Phe Arg Val Val Leu Met Tyr Arg Phe Glu Glu
325 330 335
Glu Leu Phe Leu Arg Ser Leu Gln Asp Tyr Lys Ile Gln Ser Ala Leu
340 345 350
Leu Val Pro Thr Leu Phe Ser Phe Phe Ala Lys Ser Thr Leu Ile Asp
355 360 365
Lys Tyr Asp Leu Ser Asn Leu His Glu Ile Ala Ser Gly Gly Ala Pro
370 375 380
Leu Ser Lys Glu Val Gly Glu Ala Val Ala Lys Arg Phe His Leu Pro
385 390 395 400
Gly Ile Arg Gln Gly Tyr Gly Leu Thr Glu Thr Thr Ser Ala Ile Leu
405 410 415
Ile Thr Pro Glu Gly Asp Asp Lys Pro Gly Ala Val Gly Lys Val Val
420 425 430
Pro Phe Phe Glu Ala Lys Val Val Asp Leu Asp Thr Gly Lys Thr Leu
435 440 445
Gly Val Asn Gln Arg Gly Glu Leu Cys Val Arg Gly Pro Met Ile Met
450 455 460
Ser Gly Tyr Val Asn Asn Pro Glu Ala Thr Asn Ala Leu Ile Asp Lys
465 470 475 480
Asp Gly Trp Leu His Ser Gly Asp Ile Ala Tyr Trp Asp Glu Asp Glu
485 490 495
His Phe Phe Ile Val Asp Arg Leu Lys Ser Leu Ile Lys Tyr Lys Gly
500 505 510
Tyr Gln Val Ala Pro Ala Glu Leu Glu Ser Ile Leu Leu Gln His Pro
515 520 525
Asn Ile Phe Asp Ala Gly Val Ala Gly Leu Pro Asp Asp Asp Ala Gly
530 535 540
Glu Leu Pro Ala Ala Val Val Val Leu Glu His Gly Lys Thr Met Thr
545 550 555 560
Glu Lys Glu Ile Val Asp Tyr Val Ala Ser Gln Val Thr Thr Ala Lys
565 570 575
Lys Leu Arg Gly Gly Val Val Phe Val Asp Glu Val Pro Lys Gly Leu
580 585 590
Thr Gly Lys Leu Asp Ala Arg Lys Ile Arg Glu Ile Leu Ile Lys Ala
595 600 605
Lys Lys Gly Gly Lys
610
<210> 24
<211> 535
<212> PRT
<213>Artificial sequence
<220>
<223> DED-HRV3C
<400> 24
Met Asp Tyr Lys Asp Asp Asp Asp Lys Gly Met Ala Ser Asp Leu Ser
1 5 10 15
Asn Val Glu Gly Lys Thr Gly Asn Ala Thr Asp Glu Glu Glu Glu Glu
20 25 30
Glu Glu Glu Glu Glu Glu Glu Asp Asp Asp Asp Asp Asp Asp Asp Asp
35 40 45
Asp Asp Asp Glu Asp Ser Gly Ala Glu Ile Gln Asp Asp Asp Glu Glu
50 55 60
Gly Phe Asp Asp Glu Glu Glu Phe Asp Asp Asp Asp Asp Asp Glu His
65 70 75 80
Asp Asp Asp Asp Leu Glu Asn Glu Glu Asn Glu Leu Glu Glu Leu Glu
85 90 95
Glu Arg Val Glu Ala Arg Lys Lys Met His His His His His His Ser
100 105 110
Ser Gly Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val
115 120 125
Gln Pro Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu
130 135 140
His Leu Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe
145 150 155 160
Glu Leu Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp
165 170 175
Val Lys Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys
180 185 190
His Asn Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met
195 200 205
Leu Glu Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala
210 215 220
Tyr Ser Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu
225 230 235 240
Pro Glu Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr
245 250 255
Leu Asn Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala
260 265 270
Leu Asp Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro
275 280 285
Lys Leu Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp
290 295 300
Lys Tyr Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp
305 310 315 320
Gln Ala Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val
325 330 335
Pro Arg Gly Ser Pro Glu Phe Pro Gly Arg Leu Glu Arg Pro His Arg
340 345 350
Asp Gly Pro Asn Thr Glu Phe Ala Leu Ser Leu Leu Arg Lys Asn Ile
355 360 365
Met Thr Ile Thr Thr Ser Lys Gly Glu Phe Thr Gly Leu Gly Ile His
370 375 380
Asp Arg Val Cys Val Ile Pro Thr His Ala Gln Pro Gly Asp Asp Val
385 390 395 400
Leu Val Asn Gly Gln Lys Ile Arg Val Lys Asp Lys Tyr Lys Leu Val
405 410 415
Asp Pro Glu Asn Ile Asn Leu Glu Leu Thr Val Leu Thr Leu Asp Arg
420 425 430
Asn Glu Lys Phe Arg Asp Ile Arg Gly Phe Ile Ser Glu Asp Leu Glu
435 440 445
Gly Val Asp Ala Thr Leu Val Val His Ser Asn Asn Phe Thr Asn Thr
450 455 460
Ile Leu Glu Val Gly Pro Val Thr Met Ala Gly Leu Ile Asn Leu Ser
465 470 475 480
Ser Thr Pro Thr Asn Arg Met Ile Arg Tyr Asp Tyr Ala Thr Lys Thr
485 490 495
Gly Gln Cys Gly Gly Val Leu Cys Ala Thr Gly Lys Ile Phe Gly Ile
500 505 510
His Val Gly Gly Asn Gly Arg Gln Gly Phe Ser Ala Gln Leu Lys Lys
515 520 525
Gln Tyr Phe Val Glu Lys Gln
530 535
<210> 25
<211> 78
<212> DNA
<213>Artificial sequence
<220>
<223> DE12
<400> 25
gatgacaaag gtatggctag cgaggaggaa gaggaagaag atgatgatga tgacgacgct 60
agcctggaag ttctgttc 78
<210> 26
<211> 78
<212> DNA
<213>Artificial sequence
<220>
<223> DE12
<400> 26
gaacagaact tccaggctag cgtcgtcatc atcatcatct tcttcctctt cctcctcgct 60
agccatacct ttgtcatc 78
<210> 27
<211> 30
<212> DNA
<213>Artificial sequence
<220>
<223>Primer 1
<400> 27
cgatacaaat ggaaataata accatctcgc 30
<210> 28
<211> 30
<212> DNA
<213>Artificial sequence
<220>
<223>Primer 2
<400> 28
cgcacagaat ctaacgctta ataaatgtac 30
<210> 29
<211> 825
<212> DNA
<213>Artificial sequence
<220>
<223> NQ01
<400> 29
atggtgggac gccgtgctct gatcgtgctc gctcactcgg aaagaacatc gttcaactac 60
gctatgaagg aggctgctgc cgctgccctg aagaagaagg gctgggaggt ggtcgaatcc 120
gacttgtacg ctatgaactt caaccccatc atctctcgta aggacatcac cggcaagctg 180
aaggatccag ccaacttcca gtacccggct gagtcagttt tggcctacaa ggaaggccac 240
ctgtcgcctg acatcgtggc tgagcaaaag aagctcgaag ctgccgattt ggttatcttc 300
cagttcccct tgcaatggtt cggtgtgcct gctatcctga agggctggtt cgagagggtc 360
ttcatcggag aattcgccta cacttacgct gccatgtacg acaagggtcc attcagatcg 420
aagaaggccg tcctgtccat caccactggt ggcagcggat caatgtacag cctccaggga 480
atccacggtg acatgaacgt catcctgtgg ccgatccaat ctggcatcct ccacttctgc 540
ggattccagg tgttggagcc acaactgaca tactccatcg gacacacccc agctgacgct 600
cgtatccaga tcctcgaagg atggaagaag cgcttggaga acatctggga cgaaactccc 660
ttgtacttcg ctccttcctc tctgttcgat ctcaacttcc aggccggttt cctcatgaag 720
aaggaggtcc aagacgagga aaagaacaag aagttcggcc tgtctgttgg acaccacctc 780
ggcaagagca tccccacaga taaccagatc aaggctagga agtaa 825
<210> 30
<211> 30
<212> DNA
<213>Artificial sequence
<220>
<223>Primer 3F
<400> 30
ctgttccagg gtcccatggt gggacgccgt 30
<210> 31
<211> 36
<212> DNA
<213>Artificial sequence
<220>
<223>Primer 3R
<400> 31
ggagctcgaa ttcccttact tcctagcctt gatctg 36
<210> 32
<211> 96
<212> DNA
<213>Artificial sequence
<220>
<223> DE18
<400> 32
gatgacaaag gtatggctag cgaagaagag gaggaggaag aggaagaaga tgatgatgat 60
gacgacgacg acgacgctag cctggaagtt ctgttc 96
<210> 33
<211> 96
<212> DNA
<213>Artificial sequence
<220>
<223> DE18
<400> 33
gaacagaact tccaggctag cgtcgtcgtc gtcgtcatca tcatcatctt cttcctcttc 60
ctcctcctct tcttcgctag ccataccttt gtcatc 96
<210> 34
<211> 273
<212> DNA
<213>Artificial sequence
<220>
<223> DED
<400> 34
gatctaagta atgtggaagg taagacagga aatgcaacag atgaagagga ggaagaagag 60
gaggaggaag aggaagaaga tgatgatgat gacgacgacg acgacgatga tgatgaagac 120
tctggagctg agatacaaga tgatgatgag gaaggttttg atgatgaaga ggaatttgat 180
gatgacgatg atgatgaaca tgatgatgat gatcttgaga atgaggaaaa cgaactggaa 240
gagttggaag agagggtaga agccaggaag aaa 273
<210> 35
<211> 34
<212> DNA
<213>Artificial sequence
<220>
<223>Primer 4F
<400> 35
aatagatctt ggtacccatg gaagaggagg aaga 34
<210> 36
<211> 63
<212> DNA
<213>Artificial sequence
<220>
<223>Primer 4R
<400> 36
ggagctcgaa ttcccgggac cctggaacag aacttccagg gtacctttat catcatcgtc 60
gtc 63
<210> 37
<211> 132
<212> DNA
<213>Artificial sequence
<220>
<223> DE30
<400> 37
gaacagaact tccaggctag catcatcatc gtcgtcgtcg tcgtcatcat catcatcgtc 60
gtcgtcctct tcctcttctt cctcttcctc ctcctcttct tcctcctctt cgctagccat 120
acctttgtca tc 132
<210> 38
<211> 132
<212> DNA
<213>Artificial sequence
<220>
<223> DE30
<400> 38
gatgacaaag gtatggctag cgaagaggag gaagaagagg aggaggaaga ggaagaagag 60
gaagaggacg acgacgatga tgatgatgac gacgacgacg acgatgatga tgctagcctg 120
gaagttctgt tc 132
<210> 39
<211> 150
<212> DNA
<213>Artificial sequence
<220>
<223> DE36
<400> 39
gaacagaact tccaggctag catcatcatc gtcgtcgtcg tcgtcatcat catcatcgtc 60
gtcgtcgtcg tcgtcctctt cctcctcttc ctcttcttcc tcttcctcct cctcttcttc 120
ctcctcttcg ctagccatac ctttgtcatc 150
<210> 40
<211> 150
<212> DNA
<213>Artificial sequence
<220>
<223> DE36
<400> 40
gatgacaaag gtatggctag cgaagaggag gaagaagagg aggaggaaga ggaagaagag 60
gaagaggagg aagaggacga cgacgacgac gacgatgatg atgatgacga cgacgacgac 120
gatgatgatg ctagcctgga agttctgttc 150
<210> 41
<211> 36
<212> DNA
<213>Artificial sequence
<220>
<223>Primer 5F
<400> 41
caaaggtatg gctagcgatc taagtaatgt ggaagg 36
<210> 42
<211> 35
<212> DNA
<213>Artificial sequence
<220>
<223>Primer 5R
<400> 42
gaacttccag gctagctttc ttcctggctt ctacc 35
<210> 43
<211> 38
<212> DNA
<213>Artificial sequence
<220>
<223>Primer 6F
<400> 43
gacaaaggta tggctagcga tctaagtaat gtggaagg 38
<210> 44
<211> 37
<212> DNA
<213>Artificial sequence
<220>
<223>Primer 6R
<400> 44
ggaacagaac ttccaggcta gcctcagctc cagagtc 37
<210> 45
<211> 114
<212> DNA
<213>Artificial sequence
<220>
<223> EO24
<400> 45
gatgacaaag gtatggctag cgaagaggag gaagaagagg aggaggaaga ggaagaagaa 60
gaggaggaag aagaggagga ggaagaggaa gaagctagcc tggaagttct gttc 114
<210> 46
<211> 114
<212> DNA
<213>Artificial sequence
<220>
<223> EO24
<400> 46
gaacagaact tccaggctag cttcttcctc ttcctcctcc tcttcttcct cctcttcttc 60
ttcctcttcc tcctcctctt cttcctcctc ttcgctagcc atacctttgt catc 114
<210> 47
<211> 35
<212> DNA
<213>Artificial sequence
<220>
<223>Primer 7F
<400> 47
gttccagggt ccagctagcg atctaagtaa tgtgg 35
<210> 48
<211> 34
<212> DNA
<213>Artificial sequence
<220>
<223>Primer 7R
<400> 48
cgtccttgta gtcgctagct ttcttcctgg cttc 34
<210> 49
<211> 33
<212> DNA
<213>Artificial sequence
<220>
<223>Primer 8F
<400> 49
cctataaata gatctcccat ggtgggacgc cgt 33
<210> 50
<211> 36
<212> DNA
<213>Artificial sequence
<220>
<223>Primer 8R
<400> 50
gaacagaact tccagcccct tcctagcctt gatctg 36
<210> 51
<211> 39
<212> DNA
<213>Artificial sequence
<220>
<223>Primer 9F
<400> 51
gttccagggt ccagctagcg atctaagtaa tgtggaagg 39
<210> 52
<211> 37
<212> DNA
<213>Artificial sequence
<220>
<223>Primer 9R
<400> 52
catcgtcctt gtagtcgcta gcctcagctc cagagtc 37
<210> 53
<211> 1638
<212> DNA
<213>Artificial sequence
<220>
<223>Luciferase
<400> 53
gaagacgcca aaaacataaa gaaaggcccg gcgccattct atcctctaga ggatggaacc 60
gctggagagc aactgcataa ggctatgaag agatacgccc tggttcctgg aacaattgct 120
tttacagatg cacatatcga ggtgaacatc acgtacgcgg aatacttcga aatgtccgtt 180
cggttggcag aagctatgaa acgatatggg ctgaatacaa atcacagaat cgtcgtatgc 240
agtgaaaact ctcttcaatt ctttatgccg gtgttgggcg cgttatttat cggagttgca 300
gttgcgcccg cgaacgacat ttataatgaa cgtgaattgc tcaacagtat gaacatttcg 360
cagcctaccg tagtgtttgt ttccaaaaag gggttgcaaa aaattttgaa cgtgcaaaaa 420
aaattaccaa taatccagaa aattattatc atggattcta aaacggatta ccagggattt 480
cagtcgatgt acacgttcgt cacatctcat ctacctcccg gttttaatga atacgatttt 540
gtaccagagt cctttgatcg tgacaaaaca attgcactga taatgaattc ctctggatct 600
actgggttac ctaagggtgt ggcccttccg catagaactg cctgcgtcag attctcgcat 660
gccagagatc ctatttttgg caatcaaatc attccggata ctgcgatttt aagtgttgtt 720
ccattccatc acggttttgg aatgtttact acactcggat atttgatatg tggatttcga 780
gtcgtcttaa tgtatagatt tgaagaagag ctgtttttac gatcccttca ggattacaaa 840
attcaaagtg cgttgctagt accaacccta ttttcattct tcgccaaaag cactctgatt 900
gacaaatacg atttatctaa tttacacgaa attgcttctg ggggcgcacc tctttcgaaa 960
gaagtcgggg aagcggttgc aaaacgcttc catcttccag ggatacgaca aggatatggg 1020
ctcactgaga ctacatcagc tattctgatt acacccgagg gggatgataa accgggcgcg 1080
gtcggtaaag ttgttccatt ttttgaagcg aaggttgtgg atctggatac cgggaaaacg 1140
ctgggcgtta atcagagagg cgaattatgt gtcagaggac ctatgattat gtccggttat 1200
gtaaacaatc cggaagcgac caacgccttg attgacaagg atggatggct acattctgga 1260
gacatagctt actgggacga agacgaacac ttcttcatag ttgaccgctt gaagtcttta 1320
attaaataca aaggatatca ggtggccccc gctgaattgg aatcgatatt gttacaacac 1380
cccaacatct tcgacgcggg cgtggcaggt cttcccgacg atgacgccgg tgaacttccc 1440
gccgccgttg ttgttttgga gcacggaaag acgatgacgg aaaaagagat cgtggattac 1500
gtcgccagtc aagtaacaac cgcgaaaaag ttgcgcggag gagttgtgtt tgtggacgaa 1560
gtaccgaaag gtcttaccgg aaaactcgac gcaagaaaaa tcagagagat cctcataaag 1620
gccaagaagg gcggaaag 1638
<210> 54
<211> 30
<212> DNA
<213>Artificial sequence
<220>
<223>Primer 10F
<400> 54
ctgttccagg gtcccgaaga cgccaaaaac 30
<210> 55
<211> 35
<212> DNA
<213>Artificial sequence
<220>
<223>Primer 10R
<400> 55
ggagctcgaa ttcccttact ttccgccctt cttgg 35
<210> 56
<211> 1296
<212> DNA
<213>Artificial sequence
<220>
<223> HRV3C
<400> 56
atgcaccacc accatcatca ttcgagcggt atgagcccca ttttggggta ttggaaaatc 60
aaaggtctgg ttcaaccaac ccggctcctg cttgaatatc ttgaagagaa atacgaagag 120
catctgtatg aacgtgacga aggcgataaa tggcgcaata agaagtttga acttggcctg 180
gagtttccga acttgccgta ttacattgat ggcgatgtga aactgacaca gtctatggcg 240
attattcgct atattgcgga caaacacaac atgttaggcg gttgcccgaa agaacgtgcg 300
gaaatctcaa tgttagaagg ggctgttctc gatattcgct atggcgtgtc tcgtatcgca 360
tacagtaaag actttgaaac gctgaaagtc gattttcttt cgaaattgcc ggagatgctg 420
aaaatgttcg aagatcggtt gtgccacaaa acgtatctga acggggatca tgtcacccat 480
ccggatttca tgttgtacga tgctctggat gtggtgctgt atatggaccc aatgtgcttg 540
gacgcgtttc caaagctggt gtgtttcaag aaacgcattg aggccattcc gcagattgat 600
aaatacctga aaagctcgaa atatattgcg tggcctctgc agggttggca agccaccttt 660
ggtggcggag atcaccctcc gaaaagcgat ctggtcccgc gtgggagtcc tgaatttcca 720
ggtcgccttg agcgcccgca tcgtgatggt ccgaacacgg aattcgcact gtccctcctg 780
cgcaagaaca ttatgacaat caccacgagc aaaggcgaat tcactggact gggaatccat 840
gatcgcgtgt gtgttattcc cacccatgca cagcctggtg atgacgtcct ggtaaatggc 900
cagaaaatcc gcgttaaaga caaatacaaa ctggtagacc cggaaaacat caatctcgaa 960
ctgaccgtgt taaccttaga ccgtaacgag aaatttcgcg acattcgcgg tttcatttcc 1020
gaggacctcg aaggtgtgga tgcaacgctg gtagtgcatt ccaacaattt cacgaatacc 1080
atcctggaag ttggcccggt tacaatggcc ggcttaatca acctgtctag tactcccacc 1140
aatcgtatga ttcgctatga ttacgcgacc aagactggcc aatgtggtgg agtcttatgc 1200
gctactggca aaatctttgg gatccacgtt ggtggcaatg gccgtcaggg cttttcagcc 1260
caactgaaga aacagtactt cgtagaaaag cagtaa 1296
<210> 57
<211> 42
<212> DNA
<213>Artificial sequence
<220>
<223>Primer 11F
<400> 57
cgatgacaaa ggtatggcta gcgatctaag taatgtggaa gg 42
<210> 58
<211> 39
<212> DNA
<213>Artificial sequence
<220>
<223>Primer 11R
<400> 58
gatgatggtg gtggtgcatt ttcttcctgg cttctaccc 39
<210> 59
<211> 39
<212> DNA
<213>Artificial sequence
<220>
<223>Primer 12F
<400> 59
gggtagaagc caggaagaaa atgcaccacc accatcatc 39
<210> 60
<211> 44
<212> DNA
<213>Artificial sequence
<220>
<223>Primer 12R
<400> 60
ctcgaggagc tcgaattccc ttactgcttt tctacgaagt actg 44
<210> 61
<211> 45
<212> DNA
<213>Artificial sequence
<220>
<223>Primer 13F
<400> 61
ctttaagaag gagatataca tatggactac aaggacgacg atgac 45
<210> 62
<211> 45
<212> DNA
<213>Artificial sequence
<220>
<223>Primer 13R
<400> 62
ccaccagtca tgctagccat atgttactgc ttttctacga agtac 45
<210> 63
<211> 16
<212> DNA
<213>Artificial sequence
<220>
<223>Primer 14
<400> 63
atgcgtccgg cgtaga 16
<210> 64
<211> 18
<212> DNA
<213>Artificial sequence
<220>
<223>Primer 15
<400> 64
ccctcaagac ccgtttag 18
<210> 65
<211> 6
<212> PRT
<213>Artificial sequence
<220>
<223> DE6
<400> 65
Glu Glu Glu Asp Asp Asp
1 5
<210> 66
<211> 60
<212> DNA
<213>Artificial sequence
<220>
<223> DE6
<400> 66
gatgacaaag gtatggctag cgaggaagaa gatgatgatg ctagcctgga agttctgttc 60
<210> 67
<211> 60
<212> DNA
<213>Artificial sequence
<220>
<223> DE6
<400> 67
gaacagaact tccaggctag catcatcatc ttcttcctcg ctagccatac ctttgtcatc 60

Claims (19)

1. protein purification method, it includes：
Modulation contains the sample of the fused protein of the amino acid sequence of amino acid sequence and target protein containing peptide tag Process and
The fused protein that will be together contained in above-mentioned fused protein in scrambled proteins matter and the said sample in said sample The process of separation,
Above-mentioned peptide tag is containing acidic amino acid residues more than 12 residues.
2. the method described in claim 1, wherein above-mentioned peptide tag has SEQ ID NO：The amino acid sequence of any one of 1~8.
3. the method described in claim 1, wherein above-mentioned peptide tag is containing acidic amino acid residues more than 18 residues.
4. the method described in claim 1, wherein the isoelectric point of above-mentioned fused protein is less than 6.
5. the salinity of the method described in claim 1, wherein said sample is more than 50mM below 500mM.
6. the method described in claim 1, wherein in said sample modulating process, containing to host cell importing and encoding above-mentioned melt The carrier of the polynucleotides of hop protein matter, makes protein obtain said sample in host cell expression.
7. the method described in claim 1, wherein amino acid sequence and above-mentioned target egg of the above-mentioned fused protein in peptide tag The cut-out site also recognized between the amino acid sequence of white matter containing protein decomposition enzyme.
8. the method described in claim 1, wherein in above-mentioned separation circuit, above-mentioned fusion egg is carried out using ion exchange resin The separation of white matter and above-mentioned scrambled proteins matter.
9. the method described in claim 1, wherein above-mentioned ion exchange resin is anion exchange resin.
10. the method described in claim 1, wherein in above-mentioned separation circuit, by making said sample pass through anion exchange Resin and obtain the anion exchange resin for combining above-mentioned fused protein and containing above-mentioned scrambled proteins matter by fraction, make With more than salinity 600mM buffer solution from the above-mentioned above-mentioned fused protein of anion exchange resin dissolution.
11. the protein purification method described in any one of claim 1~10, wherein after above-mentioned separation circuit, also Including the use of the protein decomposition enzyme for recognizing above-mentioned cut-out site, above-mentioned fused protein is separated into above-mentioned target in the solution Protein and above-mentioned peptide tag, the process for obtaining above-mentioned target protein.
12. the protein purification method described in claim 11, wherein in above-mentioned target protein obtains process, making containing upper The solution of target protein and above-mentioned peptide tag is stated by anion exchange resin, the anion for combining above-mentioned peptide tag is obtained Exchanger resin and anion exchange resin containing above-mentioned target protein pass through fraction.
13. the method described in claim 10, wherein the salinity of the solution containing above-mentioned target protein and above-mentioned peptide tag is Below 500mM.
14. fused protein, the amino acid sequence of its amino acid sequence and target protein containing peptide tag, the peptide tag contains Acidic amino acid residues more than 12 residues.
15. the fused protein described in claim 14, wherein above-mentioned peptide tag has SEQ ID NO：The ammonia of any one of 1~8 Base acid sequence.
16. the fused protein described in claim 14, wherein above-mentioned peptide tag is residual containing acidic amino acids more than 18 residues Base.
17. the fused protein described in claim 14, wherein the isoelectric point of above-mentioned fused protein is less than 6.
18. the fused protein described in claim 14, wherein above-mentioned fused protein is in the amino acid sequence of peptide tag and upper State the cut-out site also recognized between the amino acid sequence of target protein containing protein decomposition enzyme.
19. the production method of the fused protein described in any one of claim 14~18, it includes：
The base sequence containing the amino acid sequence for encoding above-mentioned peptide tag is imported to host cell and encodes above-mentioned fused protein Amino acid sequence base sequence polynucleotides process and
The process that above-mentioned fused protein is expressed in above-mentioned host cell.
CN201710199777.9A 2016-04-07 2017-03-30 Protein purification method and fused protein and its production method containing peptide tag Pending CN107266523A (en)

Applications Claiming Priority (2)

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JP2016-077127 2016-04-07
JP2016077127A JP2017186277A (en) 2016-04-07 2016-04-07 Protein purification method and fusion protein comprising peptide tag and production method thereof

Publications (1)

Publication Number Publication Date
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107266526A (en) * 2016-04-07 2017-10-20 希森美康株式会社 The purification process of target protein
CN109929023A (en) * 2017-12-15 2019-06-25 中国医学科学院药物研究所 A kind of metal compatibility fusion protein label and its application
CN111072772A (en) * 2019-12-31 2020-04-28 上海良润生物医药科技有限公司 Peptide tag and application thereof in detection or purification of fusion protein

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107266526A (en) * 2016-04-07 2017-10-20 希森美康株式会社 The purification process of target protein
CN109929023A (en) * 2017-12-15 2019-06-25 中国医学科学院药物研究所 A kind of metal compatibility fusion protein label and its application
CN109929023B (en) * 2017-12-15 2022-06-21 中国医学科学院药物研究所 Metal affinity fusion protein label and application thereof
CN111072772A (en) * 2019-12-31 2020-04-28 上海良润生物医药科技有限公司 Peptide tag and application thereof in detection or purification of fusion protein
CN111072772B (en) * 2019-12-31 2023-08-04 上海良润生物医药科技有限公司 Peptide tag and application thereof in detection or purification of fusion protein

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Application publication date: 20171020